ES2715633T3 - Device and method for imaging and fluorescence monitoring - Google Patents

Abstract

A device for imaging and fluorescence monitoring of a target (10), comprising: a light source (5) that emits a light to illuminate the target, the emitted light includes at least one wavelength or longitude band wave that causes the fluorescence of at least one biomarker associated with the target; an optical filter holder (3) configured to accommodate one or more optical filters; an electrical supply (19); a digital image acquisition device (1) that has a lens (2) and is configured to acquire a fluorescence image of the target secured inside the housing, in which the digital image acquisition device is a camera digital, a video recorder, a camcorder, a mobile phone with a built-in digital camera, a smartphone with a digital camera, a personal digital assistant or a webcam; and a housing (20) that houses all the components of the device in an entity and comprises a means for fixing the digital image acquisition device in the housing, in which the housing is configured to be manual, compact and portable.

Description

DESCRIPTION

Device and method for imaging and fluorescence monitoring

Technical field

A device and method for imaging and fluorescence monitoring are disclosed. In particular, the device and method may be suitable for the monitoring of biochemical and / or biological and non-biological substances, such as wound care, for both human and animal applications.

Background

Wound care is an important clinical challenge. Healing wounds and chronic non-healing wounds are associated with a series of changes in biological tissues that include inflammation, proliferation, remodeling of connective tissues and, one of the main common concerns, bacterial infection. A proportion of wound infections are not clinically evident and contribute to the growing economic burden associated with wound care, especially in aging populations. Currently, wound evaluation by reference criteria includes a direct visual inspection of the wound site with white light combined with indiscriminate collection of bacterial smears and tissue biopsies that result in late, expensive and often bacteriological results. insensitive This may affect the timing and effectiveness of the treatment. The qualitative and subjective visual evaluation only provides an overview of the wound site, but does not provide information on the underlying biological and molecular changes that occur at the tissue and cellular level. A relatively simple and complementary method that takes advantage of "biological and molecular" information to improve the early identification of such hidden change is desirable in the clinical treatment of wounds. Early recognition of high-risk wounds can guide therapeutic intervention and provide a follow-up of the response over time, which greatly reduces morbidity and mortality due especially to chronic wounds.

Wound care and treatment is a significant clinical challenge that presents a significant burden and a challenge for medical care worldwide [Bowler et al., Clin Microbiol Rev. 2001, 14: 244-269; Cutting et al., Journal of Wound Care. 1994, 3: 198-201; Dow et al., Ostomy / Wound Management. 1999, 45: 23-40]. Wounds are generally classified as wounds without tissue loss (e.g., in surgery) and wounds with tissue loss, such as burns, wounds caused as a result of trauma, abrasions or as secondary events in chronic conditions (e.g. ., venous stasis, diabetic ulcers or pressure sores and iatrogenic wounds such as donor sites of skin grafts and dermabrasions, pilonidal sinuses, surgical wounds that do not heal and chronic cavity wounds). Wounds are also classified according to the layers involved, superficial wounds that affect only the epidermis, partial thickness wounds that affect only the epidermis and dermis, and full thickness wounds that affect subcutaneous fat or tissue deeper. Although the restoration of tissue continuity after injury is a natural phenomenon, infection, healing quality, healing speed, fluid loss and other complications that increase healing time represent a significant clinical challenge. Most wounds heal without any complications. However, chronic wounds that do not heal and progressively lead to more tissue loss are a great challenge for wound care professionals and researchers. Unlike surgical incisions where there is relatively little tissue loss and wounds usually heal without significant complications, chronic wounds alter the normal healing process, which is often not sufficient in itself to repair. Late healing is generally a result of the physiology of the compromised wound [Winter (1962) Nature. 193: 293-294] and usually occurs with venous stasis and diabetic ulcers, or prolonged local pressure as in immunosuppressed and immobilized older persons. These chronic conditions increase the cost of care and reduce the patient's quality of life. As these groups increase in number, the need for advanced wound care products will increase.

Conventional methods of clinical evaluation of acute and chronic wounds remain suboptimal. In general, they are based on a complete clinical history of the patient, a qualitative and subjective clinical evaluation with a simple visual opinion with ambient white light and with the naked eye, and sometimes they may involve the use of color photographs to capture the general appearance of a wound with white light illumination [Perednia (1991) J Am Acad Dermatol. 25: 89-108]. A regular reassessment of progress towards healing and the appropriate modification of the intervention is also necessary. The terminology of the wound assessment is not uniform, many questions surrounding the wound assessment remain unanswered, no agreement has been reached on the key parameters of the wounds to be measured in clinical practice, and the accuracy and reliability of the available wound evaluation techniques vary. Visual evaluation is often combined with sampling and / or tissue biopsies for bacteriological culture for diagnosis. Bacterial smears are collected at the time of wound examination and have the indicated advantage of providing identification of specific bacterial / microbial species [Bowler, 2001; Cutting, 1994; Dow, 1999; Dow G. in: Krasner et al. eds. Chronic Wound Care: A Clinical Source Book for Saludcare Professionals, 3rd ed. Wayne Pa .: HMP Communications. 2001: 343-356]. However, often multiple smears and / or random biopsies are collected from the wound site, and some sampling techniques they can in fact spread the microorganisms around the wound during the collection process, which affects the healing time and the morbidity of the patient [Dow, 1999]. This can be a problem, especially with large (non-healing) chronic wounds in which the detection performance of the bacterial presence through the use of current biopsy protocols and sampling is suboptimal (insensitive to diagnosis), although they are collected Many smears Therefore, current methods for obtaining smears or tissue biopsies from the wound site for subsequent bacteriological culture are based on a non-directed or "blind" sampling or puncture biopsy approach, and have not been optimized to minimize trauma to the wound or to maximize the diagnostic performance of bacteriology tests. In addition, obtaining smears and biopsy samples for bacteriology can be laborious, invasive, painful, expensive and, more importantly, the results of bacteriological cultures usually take 2 to 3 days to return from the laboratory and may not be conclusive [Serena et al. (2008) Int J Low Extrem Wounds. 7 (1): 32-5 .; Gardner et al., (2007) WOUNDS. 19 (2): 31-38], which delays accurate diagnosis and treatment [Dow, 1999]. Therefore, bacterial smears do not provide real-time detection of the infectious state of wounds. Although wound sampling seems to be simple, it can lead to inadequate treatment, patient morbidity and increased hospital stays if not performed correctly [Bowler, 2001; Cutting, 1994; Dow, 1999; Dow, 2001]. The lack of a non-invasive imaging method to objectively and quickly assess wound repair at the biological level (which may be more detailed than simple appearance or morphology), and to assist in the selective identification of the collection of Tissue smears and samples of bacterial biopsies are a major obstacle in the evaluation and treatment of clinical wounds. An alternative method is highly desirable.

As the wounds heal (chronic and acute), a series of key biological changes occur at the site of the wound at the tissue and cellular level [Cutting, 1994]. Wound healing involves a complex and dynamic interaction of biological processes divided into four overlapping phases (hemostasis, inflammation, cell proliferation and maturation or remodeling of connective tissues) that affect the pathophysiology of wound healing [Physiological basis of wound healing, in Developments in wound care, PJB Publications Ltd., 5-17, 1994]. A common major complication that arises during the wound healing process, which can vary from days to months, is an infection caused by bacteria and other microorganisms [Cutting, 1994; Dow, 1999]. This can lead to a serious impediment to the healing process and lead to significant complications. All wounds contain bacteria at levels ranging from contamination, through colonization, critical colonization to infection, and the diagnosis of bacterial infection is based on clinical signs and symptoms (e.g., visual and odorous signs ).

The most commonly used terms for wound infection have included wound contamination, wound colonization, wound infection and, more recently, critical colonization. Wound contamination refers to the presence of bacteria in a wound without any reaction from the host [Ayton M. Nurs Times 1985, 81 (46): supl. 16-19], wound colonization refers to the presence of bacteria in the wound that multiply or initiate a host reaction [Ayton, 1985], critical colonization refers to the multiplication of bacteria that cause a delay in wound healing, usually associated with an exacerbation of pain not previously notified but still without overt reaction from the host [Falanga et al., J Invest Dermatol 1994 102 (1): 125-27; Kingsley A, Nurs Stand 2001, 15 (30): 50-54, 56, 58]. Wound infection refers to the deposition and multiplication of bacteria in the tissue with an associated host reaction [Ayton, 1985]. In practice, the term "critical colonization" can be used to describe wounds that are considered to be moving from colonization to local infection. However, the challenge in the clinical setting is to ensure that this situation is quickly recognized with confidence and that the bacterial biological load is reduced as soon as possible, perhaps through the use of topical antimicrobials. Potential wound pathogens can be classified into different groups, such as bacteria, fungi, spores, protozoa and viruses, according to their structure and metabolic capacity [Cooper et al., Wound Infection and Microbiology .: Medical Communications (UK) Ltd for Johnson & Johnson Medical, 2003]. Although viruses generally do not cause wound infections, bacteria can infect skin lesions that form during the course of certain viral diseases. Such infections can occur in various settings, including in healthcare settings (hospitals, clinics) and in homes or chronic care facilities. The control of wound infections is increasingly complicated, but treatment is not always guided by microbiological diagnosis. The diversity of microorganisms and the high incidence of polymicrobial flora in most chronic and acute wounds support the value of identifying one or more bacterial pathogens from wound cultures. Early recognition of the causative agents of wound infections can help wound care professionals take appropriate measures. In addition, the formation of defective collagen is due to a greater bacterial load and results in friable loose granulation tissue with excess vascularization that generally leads to spontaneous opening of the edges of a surgical wound [Sapico et al. (1986) Diagn Microbiol Infect Dis. 5: 31-38].

Accurate and clinically relevant assessment of the wound is an important clinical tool, but this process is still an important challenge. Current visual assessment in clinical practice only provides an overview of the site of the wound (eg, presence of purulent material and scabs). Current best clinical practice does not adequately use objective information of critical importance about the underlying fundamental biological changes that occur at the tissue and cell level (e.g., contamination, colonization, infection, matrix remodeling, inflammation, bacterial infection / microbial and necrosis) since these indexes i) are not readily available at the time of wound examination and ii) are not currently integrated into the process Conventional wound treatment. The direct visual assessment of the state of health of the wound by white light is based on the detection of color and the topographic / textural changes in the wound and around it, and therefore it may be incapable and unreliable to detect changes Subtle in tissue remodeling. More importantly, direct visual evaluation of wounds often does not detect the presence of a bacterial infection, since the bacteria are hidden under white light illumination. The infection is diagnosed clinically with microbiological tests used to identify the organisms and their susceptibility to antibiotics. Although physical indications of bacterial infection can be easily observed in most wounds with white light (e.g., purulent exudate, scab formation, swelling, erythema), this is usually significantly delayed and the patient is already at greater risk. of morbidity (and other complications associated with infection) and mortality. Therefore, direct visualization with conventional white light does not detect the early presence of the bacteria themselves or identify the types of bacteria in the wound.

The implant and graft of cytoblasts have recently aroused interest, such as wound care and treatment. However, it is currently a challenge to track the proliferation of cytoblasts after implantation or grafting. The monitoring and identification of neoplastic cells has also been a challenge. It would be desirable that such cells could be monitored in a minimally invasive or non-invasive manner.

US 2008/0103355 discloses apparatus, devices, methods, systems, computer programs and calculation devices related to the formation of autofluorescent imaging and ablation.

US 5,760,407 discloses a device and a method for the identification of fluorescent follicles using a UV light fluent and a filter in front of a detector.

US 2003/0158470 discloses a device and a process for real-time detection of suspicious areas by measuring fluorescence in narrow spectral bands selected for an increase in the analysis.

It is also useful to provide a way to detect contamination of other target surfaces, including non-biological targets.

Summary

In accordance with the present invention, a device and method for fluorescence monitoring is provided as defined in the appended claims. In some aspects, the device comprises an optical device (e.g., fluorescence and / or reflectance) for non-invasive real-time imaging of biochemical and / or organic substances, for example wounds. This device can be compact, portable and / or manual, and can provide high resolution and / or high contrast images. Said device can be easily integrated into the current wound care practice. This imaging device can quickly and conveniently provide the doctor / health worker with valuable biological information of a wound: including imaging of changes in connective tissue, early detection of bacterial contamination / infection. The device can also facilitate wound margin delimitation, image-guided collection of bacterial smears / biopsy samples, imaging of activated optical contrast agents and directed by exogenous molecular biomarkers (e.g., absorption, dispersion , fluorescence, reflectance) and may allow longitudinal monitoring of a therapeutic response for adaptive intervention in the treatment of wounds. By exploiting wireless capabilities with dedicated image analysis and diagnostic algorithms, the device can be seamlessly integrated into the telemedicine infrastructure (e.g., E-health) for remote access of wound care specialists. Such a device may also have applications outside of wound care, including early detection of cancers, supervision of emerging photodynamic therapies, detection and supervision of cytoblasts, and as an instrument in dermatology and cosmetology clinics, in addition to other applications.

In accordance with the invention, a device for imaging and fluorescence monitoring of a target is provided comprising: a light source that emits a light to illuminate the target, the emitted light includes at least one wavelength or a band wavelength that causes at least one biomarker associated with the target to emit fluorescence; and a light detector to detect fluorescence.

In some examples, the device may further comprise a filter holder with the light detector, the filter holder has a plurality of optical filters that can be selected in a way that is aligned with the light detector, each optical filter is selected so that a wave is detected of length or wavelength of respective length of light.

In some examples, the target can be selected from at least one of: a surgical field, a wound, a tumor, an organ, a skin target, a biological target, a non-biological target, a food product, a plant material, a target oral, an otolaryngological target, an ocular target, a genital target, or an anal target.

In some examples, all device components can be mounted on a portable frame or on a stand

fixed.

In some examples, the device may further comprise a means or a measuring component for

Determine a distance of the device from the target. In some examples, the device may comprise the

minus two light sources at a separate fixed distance to triangulate a distance from the target device. In

Some examples, the device may comprise an ultrasound source to determine a distance from the

device from the target. In some examples, the device may comprise a physical measure to determine

a distance from the device from the target.

In some examples, the device may comprise a data port for the transmission and reception of data.

You can select from at least one of bacteria, fungi, yeasts, spores, viruses, microbes, parasites,

connective tissues, tissue components, exudates, pH, blood vessels, nicotinamide adenine dinucleotide

reduced (NADH), falvin adenine dinucleotide (FAD), microorganisms, vascular endothelial growth factor

(VEGF), endothelial growth factor (EGF), epithelial growth factor, cell membrane antigen

epithelial (ECMA), hypoxia-inducible factor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin,

fibroblast growth factor, transforming growth factors (TGF), activation protein

fibroblasts (FAP), tissue metalloproteinase inhibitors (TIMP), nitric oxide synthase (NOS), inducible NOS and

endothelial, cell lysosomes, macrophages, neutrophils, lymphocytes, hepatocyte growth factor (HGF), antineuropeptides, neutral endopeptidases (NEP), granulocyte and macrophage colony stimulating factor (GM-CSF), neutrophil elastases, cathepsins, argaseins , fibroblasts, endothelial cells and keratinocytes, factor of

keratinocyte growth (KGF), macrophage inflammatory protein-2 (MIP-2), inflammatory protein

macrophages-2 (MIP-2) and macrophage chemoattractant protein-1 (MCP-1), polymorphonuclear neutrophils (PMN),

macrophages, myofibroblasts, interleukin-1 (IL-1), tumor necrosis factor (TNF), nitric oxide (NO), c-myc, betacatenin, endothelial progenitor cells (EPCs), matrix metalloproteinases (MMPs) or m inhibitors Mp.

In some examples, the emitted light may include wavelengths or wavelength bands of

approximately 400 nm to approximately 450 nm, approximately 450 nm to approximately 500 nm, approximately 500 nm to approximately 550 nm, approximately 600 nm to approximately 650 nm, approximately 650 nm to approximately 700 nm, approximately 700 nm to approximately 750 nm, combinations of the same.

In some examples, the device may comprise a memory for recording fluorescence data from at

Less a biomarker.

In some examples, the device may comprise a processor to compare the fluorescence spectrum of

the at least one biomarker with a default query table of fluorescence spectra of

biomarkers In some aspects, a kit for image formation and supervision by

fluorescence of a target comprising: the device described above; and a contrast agent

fluorescent to mark the biomarker on the target with a wavelength or wavelength band

fluorescent detectable by the device.

In some examples, the biomarker may be a bacterium and the contrast agent may be acidic.

aminolevulinic (ALA) or PpIX.

In some examples, the contrast agent can be selected from at least one of: fluorescent dyes, dyes

chromogenic, quantum dots (QDots), molecular beacons, nanoparticles that have

fluorescent agents and dispersion or absorption nanoparticles.

In some examples, the contrast agent may include at least one moiety to target the biomarker. By

For example, the at least one residue can be selected from at least one of: antibodies, antibody fragments,

peptides, aptamers, siRNA, oligomers, receptor binding molecules, enzyme inhibitors or toxins.

In some examples, the biomarker can be selected from at least one of: bacteria, fungi, yeasts,

spores, viruses, microbes, parasites, connective tissues, tissue components, exudates, pH, blood vessels,

reduced nicotinamide adenine dinucleotide (NADH), falvin adenine dinucleotide (FAD), microorganisms,

vascular endothelial growth (VEGF), endothelial growth factor (EGF), epithelial growth factor,

epithelial cell membrane antigen (ECMA), hypoxia inducible factor (HIF-1), carbonic anhydrase IX

(CAIX), laminin, fibrin, fibronectin, fibroblast growth factor, transforming growth factors

(TGF), fibroblast activation protein (FAP), tissue metalloproteinase inhibitors (TIMP), nitric oxide

synthase (NOS), inducible and endothelial NOS, cell lysosomes, macrophages, neutrophils, lymphocytes, factor

hepatocyte growth (HGF), anti-neuropeptides, neutral endopeptidases (NEP), stimulating factor of

granulocyte and macrophage colonies (GM-CSF), neutrophil elastases, cathepsins, arginase, fibroblasts,

endothelial cells and keratinocytes, keratinocyte growth factor (KGF), inflammatory protein-2 of

macrophage (MIP-2), macrophage inflammatory protein-2 (MIP-2), macrophage chemoattractant protein-1 (MCP-1), polymorphonuclear neutrophils (PMN), macrophages, myofibroblasts, interleukin-1 (IL-1), necrosis factor tumor (TNF), nitric oxide (NO), c-myc, beta-catenin and endothelial progenitor cells (EPCs), matrix metalloproteinases (MMPs), or MMP inhibitors.

In some examples, the kit may further comprise a calibration target to measure or calibrate image parameters.

In some aspects, a method for imaging and fluorescence monitoring of a target is provided comprising: illuminating the target with a light source that emits a light of at least one wavelength or a wavelength band that makes that at least one biomarker emits fluorescence; and detect the fluorescence of the at least one biomarker with an image detector.

In some examples, the target can be selected from at least one of: a surgical field, a wound, a tumor, an organ, a skin target, a biological target, a non-biological target, a food product, a plant material, a target oral, an otolaryngological target, an ocular target, a genital target or an anal target.

In some examples, fluorescence detection may comprise detection of a biomarker fluorescence band.

In some examples, the method further comprises comparing the fluorescence band of at least one biomarker with a predetermined query table of biomarker fluorescence spectra.

In some examples, the at least one biomarker can be selected from at least one of: bacteria, fungi, yeasts, spores, viruses, microbes, parasites, connective tissues, tissue components, exudates, pH, blood vessels, reduced nicotinamide adenine dinucleotide (NADH ), falvin adenine dinucleotide (FAD), microorganisms, vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), epithelial growth factor, epithelial cell membrane antigen (ECMA), hypoxia inducible factor (HIF- 1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin, fibroblast growth factor, transforming growth factors (TGF), fibroblast activation protein (FAP), tissue metalloproteinase inhibitors (TIMP), nitric oxide synthase (NOS), inducible and endothelial NOS, cell lysosomes, macrophages, neutrophils, lymphocytes, hepatocyte growth factor (HGF), anti-neuropeptides, endopepti neutral dasas (NEP), granulocyte and macrophage colony stimulating factor (GM-CSF), neutrophil elastases, cathepsins, arginases, fibroblasts, endothelial cells and keratinocytes, keratinocyte growth factor (KGF), macrophage inflammatory protein-2 (MIP-2) and macrophage chemoattractant protein-1 (MCP-1), polymorphonuclear neutrophils (PMN), macrophages, myofibroblasts, interleukin-1 (IL-1), tumor necrosis factor (TNF), nitric oxide (NO) , c-myc, beta-catenin, endothelial progenitor cells (EPCs), matrix metalloproteinases (MMPs), or MMP inhibitors.

In some examples, the method may further comprise marking a selected biomarker on the target with at least one fluorescent contrast agent.

In some examples, the contrast agent may be aminolevulinic acid (ALA).

In some examples, the contrast agent can be selected from at least one of the fluorescent molecules, chromogenic dyes, quantum dots (QDots), molecular beacons, nanoparticles having fluorescent agents, or dispersion or absorption nanoparticles .

In some examples, the method may comprise labeling of the selected biomarker on the target with a combination of two or more contrast agents, in which the combination is specific to a selected biomarker.

In some examples, the method may further comprise providing the device or kit described above. In some examples, the method may further comprise imaging the illuminated target at separate time intervals to obtain a plurality of images of fluorescent signals from the target and evaluate the fluorescent signals from each image to determine changes in the signals fluorescent, the changes being indicative of changes in the target.

In some examples, the determined changes can be compared with known or expected changes.

In some examples, the target can be biological and the target can be evaluated to monitor the effects of therapeutic treatment over time.

In some examples, the therapeutic treatment can be selected from at least one of: pharmacological treatment, treatment with biopolymers containing drugs, debridement, photodynamic treatment, hyperbaric oxygen therapy (HOT), low level light therapy, treatment with anti-matrix metalloproteinase or treatment with a wound care product.

In some examples, the wound care product can be selected from at least one of: hydrogels, Theramers ™, gels containing silver, artificial skin, ADD cytoblasts, wet wound dressings, hydrocolloid wound dressings, transparent film dressings , antimicrobials, anti matrix metalloproteinases, dressings for active wounds or hyaluronic acid.

In some examples, the effects of therapeutic treatment can be monitored at least one of: a biological level and a physiological level.

In some examples, fluorescence detection may comprise detecting fluorescence of at least one of: a target surface and under a target surface.

In some examples, the method can be used to provide an image guide in medical or therapeutic procedures. For example, the medical or therapeutic procedure can be selected from at least one of: sampling, brushing, suction, biopsy, hyperbaric oxygen therapy, photodynamic treatment or low level light therapy.

In some examples, the method can be used in combination with an additional imaging technique. For example, the imaging technique can be selected from at least one of: thermal imaging, ultrasound, white light photography or optical devices.

In some examples, the method can be used to monitor at least one of pharmacokinetics, biodistribution and photobleaching, in PDT.

In some examples, the method can be used to detect the presence or location of a bacterial strain. For example, the bacterial strain may be at least one selected from at least one of: bacteria Staphylocuccus, Staphylococcus aureus, aeguginosa Pseudomonas, Listeria monocytogenes, Enterobacter sakazakii bacteria species Camylibacterium, coliform bacteria, Escherichia coli bacteria, Propionilbacterium acnes, or Salmonella . In some examples, the method can be used to differentiate the presence or location of two or more different bacterial strains. For example, the different bacterial strains can include Staphylococcus aureus and Pseudomonas aeguginosa , and the different bacterial strains can be differentiated based on their autofluorescence emission hallmarks.

In some examples, the method can be used to evaluate a cleaning or debridement procedure.

In some examples, the method may further comprise: storing data related to the detected fluorescence; and transmit the data to a receiving device. For example, the receiving device may be a component in a telemedicine system.

In some examples, the transmission can be performed wirelessly.

In some examples, in which the target can be a human or animal target.

In some examples, the method can be used for the detection of contamination.

In some examples, the method can be used for a direct evaluation of smears or wound smear cultures. Brief description of the drawings

Figure 1 is a schematic diagram of a device for fluorescence monitoring;

Figure 1b shows an example of a clinical wound care facility using a device for fluorescence monitoring;

Figure 2 shows images of a manual embodiment of a device for fluorescence monitoring; Figure 3 shows images of live bacterial cultures captured using a device for fluorescence monitoring;

Figure 3J shows an example of bacterial monitoring using a device for fluorescence monitoring;

Figure 4 shows images of a simulated animal wound model, demonstrating the detection of non-invasive autofluorescent bacteria using a device for fluorescence monitoring;

Figure 5 shows images of a cutaneous surface of a sample of pork, which demonstrates the non-invasive autofluorescent detection of collagen and various bacterial species using a fluorescence monitoring device;

Figure 6 shows images of a muscular surface of a sample of pork, which demonstrates the use of a fluorescence monitoring device for the detection of tissue autofluorescence. connective and bacteria;

Figure 7 shows images and spectral graphics demonstrating the use of a fluorescence monitoring device to detect the fluorescence of bacteria growing on agar plates and on the surface a simulated wound in pig meat;

Figure 8 shows images of bacterial cultures demonstrating a device for fluorescence monitoring, with and without contrast agents;

Figure 9 shows images demonstrating the use of a fluorescence monitoring device for the detection of autofluorescence of connective tissues and various bacterial species on the skin surface of a pig meat sample;

Figure 10 shows images demonstrating the use of a fluorescence monitoring device for the detection of improved bacterial infection with fluorescence contrast in a sample of pork; Figure 10G shows an example of the use of a fluorescence monitoring device to monitor the effectiveness of a photodynamic treatment;

Figure 11 shows images demonstrating the use of a fluorescence monitoring device for blood imaging and microvasculature;

Figure 12 shows images demonstrating the use of a fluorescence monitoring device for imaging the oral cavity and the skin surface;

Figure 12J shows an example of the use of a fluorescence monitoring device for imaging a skin surface;

Figure 13 shows images demonstrating the use of a fluorescence monitoring device for the detection of exogenous fluorescence contrast agents in vivo;

Figure 14 shows images demonstrating the use of a fluorescence monitoring device for fluorescence image guided surgery using image contrast agents;

Figure 15 shows images demonstrating the use of a fluorescence monitoring device for video recording of fluorescence image guided surgery;

Figure 16 shows images demonstrating the use of a fluorescence monitoring device for guided surgical resections with tissue autofluorescence imaging in a mouse heart attack model;

Figure 17 shows images demonstrating the use of a fluorescence monitoring device for autofluorescence guided image surgery of a mouse brain;

Figure 18 shows images demonstrating the use of a device for fluorescence monitoring in imaging of carcinogenic cytoblasts in a mouse;

Figure 19 shows images demonstrating the use of a device for fluorescence monitoring in the imaging of carcinogenic cytoblasts in a liver and in a lung;

Figures 19H and 19I show examples of the use of a device for fluorescence monitoring for tumor imaging;

Figure 20 shows images demonstrating the use of a device for fluorescence monitoring in imaging of a mouse model;

Figure 20B shows an example of the use of a fluorescence monitoring device for imaging small animal models;

Figure 21 illustrates the phases of wound healing over time;

Figure 22 is a table showing examples of tissue, cellular and molecular biomarkers that are known to be associated with wound healing;

Figure 23 is a diagram comparing a healthy wound with a chronic wound;

Figure 24 illustrates an example of monitoring a chronic wound;

Figures 24B-24P show examples of the use of a fluorescence monitoring device for imaging wounds and conditions in clinical patients;

Figure 24Q shows an example of the use of a device for fluorescence monitoring for imaging bacterial response to photodynamic therapy;

Figure 24R shows an example of the use of a device for fluorescence monitoring for tissue imaging;

Figure 25 is a flow chart illustrating the treatment of a chronic wound using a device for fluorescence monitoring;

Figures 26 and 27 show examples of the use of a device for fluorescence monitoring to detect contamination in food products;

Figures 28-28C show examples of the use of a device for fluorescence monitoring to detect surface contamination;

Figures 29-31 show examples of the use of a device for fluorescence monitoring for forensic applications;

Figure 32 shows an example of the use of a device for fluorescence monitoring to catalog animals;

Figure 33 shows an example of a kit that includes a device for fluorescence monitoring; and Figure 34 shows an example of the use of a fluorescence monitoring device for imaging cosmetic or dermatological substances.

Detailed description

The progression of the wound is currently monitored manually. The National Pressure Ulcer Advisory Panel (NPUAP) developed the Scale Tool for Pressure Ulcer Healing (PUSH) that describes a five-step method for characterizing pressure ulcers. This tool uses three parameters to determine a quantitative score that is then used to monitor pressure ulcer over time. Qualitative parameters include the dimensions of the wound, the type of tissue and the amount of exudate or discharge, and the thermal readings present after removing the dressing. A wound can be characterized by its smell and color. Such wound assessment does not currently include critical biological and molecular information about the wound. Therefore, all wound descriptions are somewhat subjective and are handwritten by the doctor or nurse.

What is desirable is a robust, cost-effective, non-invasive and rapid method or device based on imaging to objectively assess wounds for changes at the biological, biochemical and cellular levels and to detect quickly, sensitively and non-invasively the earlier presence of bacteria / microorganisms in wounds. Such a method or device for the detection of critical changes in biological tissues in wounds can play a complementary role with conventional clinical methods of wound treatment in order to guide key clinical-pathological decisions in patient care. Said device can be compact, portable and capable of performing non-invasive and / or non-contact wound consultations in real time in a safe and convenient manner, which may allow it to adapt perfectly to the routine practice of wound treatment and be intuitive. for doctor, nurse and wound specialist. This may also include the use of this device in the home care environment (including personal use by a patient), as well as in military battlefield environments. In addition, such an image-based device can provide an ability to monitor the wound's treatment and cure response in real time by incorporating a valuable "biologically informed" image guide into the clinical evaluation process of the wound. Ultimately, this may lead to potential new diagnoses, treatment planning, monitoring of the response to treatment and, therefore, "adaptive" intervention strategies that may allow an improvement in the wound healing response at the level of individual patient The precise identification of the systemic, local and molecular factors that underlie the problem of wound healing in individual patients may allow a better adapted treatment.

A series of imaging technologies have been made available that offer the potential to meet the requirements to improve clinical diagnosis and treatment of the disease. Of these, fluorescence imaging seems promising to improve the evaluation and treatment of the clinical wound. When excited with short wavelength light (e.g., ultraviolet or short visible wavelengths), most of the endogenous biological components of the tissues (e.g., connective tissues, such as collagen and elastins, Metabolic coenzymes, proteins, etc.) produce a fluorescence of a longer wavelength, in the ultraviolet, visible, near infrared and infrared wavelength ranges [DaCosta et al., Photochem Photobiol. October 2003, 78 (4): 384-92]. Tissue autofluorescence imaging, the most clinically mature imaging technology of emerging optic-based imaging, has been used to improve endoscopic detection of early cancers and other diseases in the gastrointestinal tract [Dacosta (2002) J Gastroenterol Hepatol. Suppl .: S85-104], the oral cavity [Poh et al., Head Neck. January 2007, 29 (1): 71-6], and lungs [Hanibuchi et al., (2007) J Med. Invest. 54: 261-6] and bladder [D'Hallewin et al. (2002) Eur Urol.

42 (5): 417-25] in a minimally invasive manner.

Tissue autofluorescence imaging provides a unique means of obtaining biologically relevant information from normal and diseased tissues in real time, allowing differentiation between normal and diseased tissue states [DaCosta, 2003; DaCosta et al. J Clin Pathol. 2005, 58 (7): 766-74]. This is based, in part, on the intrinsically different interactions between light and tissue (e.g., absorption and light scattering) that occur in mass tissue and cell levels, changes in tissue morphology and alterations in the blood content of tissues. In tissues, blood is an important component of tissue that absorbs light (that is, a chromophore). This type of technology is suitable for imaging of diseases in hollow organs (e.g., GI tract, oral cavity, lungs, bladder) or exposed tissue surfaces (e.g., skin). Despite this indication, current endoscopic fluorescence imaging systems are large, complex and expensive diagnostic algorithms are involved and, to date, these instruments are mainly in large clinical centers and very few systems are commercially available. Currently, there is no such optical imaging or fluorescence device for wound imaging. However, since the wounds are easily accessible, an autofluorescence imaging device can be useful for rapid, non-invasive and non-real-time imaging of the wounds, to detect and exploit the rich biological information of the wound to overcome current limitations and improve clinical care and treatment.

A method and a device for imaging and fluorescence monitoring are disclosed. An embodiment of the device is a portable optical digital imaging device. The device can use a combination of white light, tissue fluorescence and reflectance imaging, and can provide real-time wound imaging, evaluation, registration / documentation, supervision and / or care management. The device can manual, compact and / or lightweight. This device and this method may be suitable for wound monitoring in humans and animals.

Other uses for the device may include:

• Formation of clinically based images and research on small and large animals (eg, veterinary).

• Detection and monitoring of contamination (eg, bacterial contamination) in the preparation of food / animal products in the meat, poultry, dairy, fisheries, agricultural industries.

• Detection of "surface contamination" (eg, bacterial or biological contamination) in public (eg, medical care) and private environments.

• Multispectral imaging and detection of cancers in human and / or veterinary patients.

• As a research tool for multispectral imaging and cancer monitoring in experimental animal models of human diseases (eg, wounds and cancers).

• Forensic detection, for example, latent fingerprints and biological liquids on non-biological surfaces. • Image formation and supervision of dental plaques, excipients and cancers in the oral cavity.

• Device for imaging and supervision in clinical microbiological laboratories.

• Antibacterial tests (eg, antibiotics), disinfectant agents.

The device may generally comprise: i) one or more sources of excitation / illumination light and ii) a detector device (e.g., a digital imaging device), which can be combined with one or more filters emission optics, or spectral filtering mechanisms, and which may have a display / control screen (e.g., a touch screen), image capture and zoom controls. The device may also have: iii) a wired and / or wireless data transfer port / module, iv) an electrical power source and power / control switches, and / or v) a protective case, which can be compact and / or light, and which may have a mechanism for fixing the detector device and / or a handle. The excitation / illumination light sources can be LED arrays that emit light at approximately 405 nm (e.g., / - 5 nm), and can be coupled with additional bandpass filters centered at approximately 405 nm to eliminate / minimize the lateral spectral bands of the LED matrix output light so as not to cause light leakage in the imaging detector with its own optical filters. The digital imaging device can be a digital camera, for example, with at least an ISO800 sensitivity, but more preferably an ISO3200 sensitivity, and can be combined with one or more optical emission filters or other mechanized spectral filtering mechanisms likewise effective (e.g. miniaturized) (e.g. acoustic-optical tunable filter or tunable liquid crystal filter). The digital imaging detector device may have a display and / or touch control screen, image capture and zoom controls. The protective case can be an outer shell of hard plastic or polymer, which contains inside the digital imaging detector device, with buttons such that the user can access all the necessary controls of the device and easily manipulate them. Miniature heat sinks or small mechanical fans, or other heat dissipation devices can be inserted into the device to allow excess heat to be removed from excitation light sources, if necessary. The complete device, including all its accessories and integrated accessory parts, can be powered by conventional AC / DC power and / or a rechargeable battery pack. The complete device can also be fixed or mounted to an external mechanical device (e.g., a tripod or a mobile stand with a swivel arm) that allows mobility of the device in a clinical room with the operation of the hands-free device. Alternatively, the device may be provided with a mobile frame such that it is portable. The device can be cleaned with a wet gauze dipped in water, while the handle can be cleaned with a wet gauze dipped in alcohol. The device may include software that allows a user to control the device, including control of image formation parameters, image display, storage of image data and user information, transfer of images and / or data associated, and / or analysis of relevant images (e.g., diagnostic algorithms).

A schematic diagram of an example of the device is shown in Figure 1. The device is shown positioned to visualize a target object 10 or target surface. In the example shown, the device has a digital image acquisition device 1, such as a digital camera, video recorder, camcorder, mobile phone with built-in digital camera, a "smart phone" with digital camera, personal digital assistant (PDA), laptop / PC with a digital camera or a webcam. The digital image acquisition device 1 has an objective 2, which can be aligned to target the target object 10 and can detect the optical signal emanating from the object 10 or the surface. The device has an optical filter holder 3 that can accommodate one or more optical filters 4. Each optical filter 4 can have different spectral bandwidths and can be bandpass filters. These optical filters 4 can be selected and moved from the objective of the digital camera to selectively detect specific optical signals based on the wavelength of the light. The device may include light sources 5 that produce excitation light to illuminate the object 10 in order to obtain an optical signal (e.g., fluorescence) to obtain images, for example, with blue light (e.g., 400-450 nm), or any other combination of single or multiple wavelengths (e.g., wavelengths in the ultraviolet / visible / near infrared / infrared ranges). The light source 5 may comprise an array of LEDs, laser diodes and / or filtered lights arranged in a variety of geometries. The device may include a method or apparatus 6 (eg, a heatsink or a cooling fan) for dissipating heat and cooling lighting sources 5. The device may include a method or apparatus 7 (e.g. e.g., an optical bandpass filter) to eliminate any unwanted wavelength of light from the light sources 5 used to illuminate the object 10 that It is being represented by images. The device may include a method or apparatus 8 for using an optical medium (e.g., the use of compact miniature laser diodes that emit a collimated beam of light) to measure and determine the distance between the imaging device and object 10. For example, the device can use two light sources, such as two laser diodes, as part of a triangulation apparatus to maintain a constant distance between the device and object 10. Other light sources may be possible. The device can also use ultrasound, or a physical measurement, as a rule, to determine a constant distance to maintain. The device may also include a method or apparatus 9 (e.g., a pivot) to allow manipulation and orientation of the excitation light sources 5, 8 to maneuver these sources 5.8 to change the lighting angle of the light that strikes object 10 for variable distances.

The target object 10 can be marked with a mark 11 to allow multiple images of the object to be taken and then recorded together for analysis. The mark 11 may involve, for example, the use of exogenous fluorescence dyes of different colors that can produce multiple different optical signals when illuminated by the light sources 5 and be detectable in the image of the object 10 and therefore may allow the orientation of multiple images (e.g., taken over time) of the same region of interest by registering together the different colors and distances between them. The digital image acquisition device 1 may include one or more of: an interface 12 for a viewing helmet; an interface 13 for an external printer; an interface 14 for a tablet, a laptop, a desktop computer or other computing device; an interface 15 for the device to allow wired or wireless transfer of image data to a remote site or other device; an interface 16 for a global positioning system (GPS) device; an interface 17 for a device that allows the use of additional memory; and an interface 18 for a microphone.

The device includes an electrical supply 19 such as an AC / DC electrical supply, a compact battery bank or a rechargeable battery. Alternatively, the device can be adapted to connect to an external supply source. The device may have a housing 20 that houses all the components in an entity. The housing 20 may be equipped with a means to secure any digital imaging device therein. The housing 20 may be designed to be manual, compact and / or portable. The housing 20 may have one or more protective boxes.

With reference still to Figure 1, b) shows an example of the device in a typical wound care facility. a) shows a typical clinical wound care facility, which shows the examination chair and the accessory table. b-c) an example of the device is shown in its hardened layer container. The device can be integrated into the usual practice of wound care, which allows to obtain images of the patient in real time. d) an example of the device (arrow) placed in the "wound care cart" is shown to illustrate the size of the device. e) the device can be used to obtain images with white light illumination, while f) shows the device being used to take fluorescence images of a wound under dim lights. g) the device can be used in telemedicine / telehealth infrastructures, for example, fluorescence images of a patient's wounds can be emailed to a wound care specialist through a wireless communication device, such as a smartphone in another hospital, using a wireless / WiFi internet connection. Using this device, high resolution fluorescence images can be sent as email attachments to wound care specialists from remote wound care sites for immediate consultation with clinical experts, microbiologists, etc. in specialized centers of care and treatment of clinical wounds.

Examples

An example of a device for fluorescence monitoring is described below. All examples are provided for illustrative purposes only and are not intended to be limiting. Parameters such as wavelengths, dimensions and incubation time described in the examples can be approximate and are provided only as examples.

In this example, the devices use two LED arrays of blue / violet light (e.g., 405 nm / -10 nm emission, narrow emission spectrum) (Opto Diode Corporation, Newbury Park, California), each located on both sides of the mounting of the imaging detector as sources of excitation light or illumination. These matrices have an output power of approximately 1 watt each, which emanates from 2.5 x 2.5 cm2, with a beam angle of 70 degrees. LED arrays can be used to illuminate the tissue surface from a distance of approximately 10 cm, which means that the total optical power density on the skin surface is approximately 0.08 W / cm2. At such low powers, no potential damage is known to the target wound or to the surface of the skin, or to the eyes caused by the excitation light. However, it may not be advisable to aim the light directly at any person's eyes during imaging procedures. It should also be taken into account that the 405 nm light does not represent a health risk in accordance with international standards formulated by the International Electrotechnical Commission (IEC), as detailed on the website: http: //www.iec .ch / online_news / etech / arch_2006 / etech_0906 / focus.htm.

The one or more light sources can be articulated (e.g., manually) to vary the angle of illumination and the size of the point on the surface of the image, for example, using a built-in pivot, and are fed, by for example, through an electrical connection to an electrical outlet and / or a set of separate portable rechargeable batteries. The excitation / illumination light can be produced by sources that include, among others, single or multiple light emitting diodes (LEDs) in any arrangement, including ring or matrix formats, light-wave filtered light bulbs or lasers Individual and multiple selected excitation / illumination light sources with specific wavelength characteristics at ultraviolet (UV), visible (VIS), far infrared, near infrared (NIR) and infrared (IR) ranges can also be used, and they can be composed of a matrix of LED, organic LED, laser diode or filtered lights arranged in a variety of geometries. The excitation / illumination light sources can be "tuned" to allow the intensity of the light emanating from the device to adjust while images are being formed. The intensity of the light can be variable. LED arrays can be attached to individual cooling fans or heat sinks to dissipate the heat produced during operation. LED arrays can emit a narrow light of 405 nm, which can be spectrally filtered using a commercially available bandpass filter (Chroma Technology Corp, Rockingham, VT, USA) to reduce the potential "leakage" of the light emitted to the detector optics. When the device is held on a tissue surface (e.g., a wound) to obtain images, the light sources that illuminate can make a wavelength of bandwidth or broadband violet / blue or another wavelength or wavelength band of light on the tissue / wound surface thus producing a flat and homogeneous field in the region of interest. The light can also illuminate or excite the tissue to a certain surface depth. This excitation / illumination light interacts with normal and diseased tissues and can cause an optical signal (eg, absorption, fluorescence and / or reflectance) to be generated in the tissue.

By changing the excitation and emission wavelengths accordingly, the imaging device can interrogate tissue components (e.g., connective tissues and bacteria in a wound) on the surface and at certain tissue depths ( e.g. a wound). For example, by changing from a light of violet / blue wavelength] 400-500 nm) to green] 500-540 nm), the excitation of fluorescent sources of deeper tissues / bacteria can be achieved, for example in a wound . Similarly, by detecting longer wavelengths, fluorescence emission from deeper tissue and / or bacterial sources in the tissue can be detected on the surface of the tissue. For the evaluation of wounds, the ability to interrogate the fluorescence of the surface and / or the subsurface may be useful, for example, in the detection and possible identification of bacterial contamination, colonization, critical colonization and / or infection, which may occur in the surface and often in depth in a wound (eg, in chronic wounds that do not heal). In one example, with reference to Figure 6, c) the detection of bacteria is shown beneath the surface of the skin (i.e., in depth) after wound cleaning. This use of the device to detect bacteria on the surface and in depth in a wound and in the surrounding tissue can be evaluated in the context of other clinical signs and symptoms conventionally used in wound care centers.

Exemplary embodiments of the device are shown in Figure 2. The device can be used with any conventional compact digital imaging device (eg, a coupled charge device (CCD) or complementary metal semiconductor sensors). oxide (CMOS)) as image acquisition devices. The exemplary device shown in a) has an external power supply, the two LED arrays for illuminating the object / surface to be photographed, and a commercially available digital camera firmly attached to a lightweight metal frame equipped with A handy handle for imaging. A multiband filter is held in front of the digital camera to allow wavelength filtering of the detected optical signal emanating from the object / surface being photographed. The camera's video / USB output cables allow the transfer of image data to a computer for storage and further analysis. This example uses a commercially available Sony 8.1 megapixel digital camera (Sony Cybershot DSC-T200 digital camera, Sony Corporation, North America). This camera may be suitable due to i) its slim vertical design that can be easily integrated into the frame of the protective case, ii) its large 3.5-inch widescreen LCD touch screen for easy control, iii) its purpose of Carl Zeiss 5x optical zoom and iv) use in low light (e.g., ISO 3200). The device may have a built-in flash that allows you to obtain conventional white light images (e.g., high-definition still images or video with sound recording output). The camera's interface ports can support the transfer of wired (e.g., USB) or wireless data (e.g., Bluetooth, WiFi and similar modes) or additional third-party modules to a variety of external devices, such such as: visor helmet, an external printer, a tablet, a laptop, a desktop personal computer, a wireless device to allow the transfer of image data to a remote site / other device, a device with a global positioning system (GPS ), a device that allows the use of extra memory, and a microphone. The digital camera works with rechargeable batteries or AC / DC power. The digital imaging device may include, but is not limited to, digital cameras, webcams, digital SLR cameras, camcorders / video recorders, mobile phones with integrated digital cameras, Smartphones ™, personal digital assistants (PDAs) and laptops / tablets , or personal desktops, all of which contain / or are connected to a digital image detector / sensor.

This light signal produced by the excitation / illumination light sources can be detected by the imaging device using optical filters (e.g., those available from Chroma Technology Corp, Rockingham, VT, USA) that they reject the excitation light but allow to detect the selected wavelengths of the light emitted from the tissue, thus forming an image on the screen. There is an optical filter holder fixed to the frame of the protective case in front of the lens of the digital camera that can accommodate one or more optical filters with different widths of discrete spectral band, as shown in b) and c) of Figure 2. b) shows the device with the LED arrays on to emit a bright violet / blue light, with a single emission filter in place. c) show the device using a multiple optical filter holder used to select the appropriate filter for specific imaging of the desired wavelength, d) show the device that is held with one hand while taking pictures of the skin surface of one foot.

These bandpass filters can be selected and aligned in front of the digital camera lens to selectively detect specific optical signals from the tissue / wound surface depending on the wavelength of the desired light. Spectral filtering of the detected optical signal (e.g., absorption, fluorescence, reflectance) can also be achieved, for example, using a tunable liquid crystal filter (LCTF), or an acoustic-optical tunable filter (AOTF) which It is an electronically tunable spectral bandpass filter in solid state. Spectral filtering may also involve the use of continuous variable filters and / or optical filters of manual bandpass. These devices can be placed in front of the imaging detector to produce multispectral, hyperspectral images and / or selective wavelengths of tissues.

The device can be modified using optical polarization or variable orientation filters (e.g. linear or circular combined with the use of optical wave plates) reasonably fixed to the excitation / illumination light sources and to the detection device imaging In this way, the device can be used to obtain tissue surface images with polarized light illumination and non-polarized light detection or vice versa, or polarized light illumination and polarized light detection, either with white light reflectance and / or fluorescence images. This may allow obtaining images of wounds with minimized mirror reflections (e.g., white light image reflections), as well as allowing fluorescence polarization images and / or anisotropy-dependent changes in connective tissues (e.g., collagens and elastin) in the wound and surrounding normal tissues. This may provide useful information about the spatial orientation and organization of connective tissue fibers associated with wound remodeling during healing [Yasui et al., (2004) Appl. To opt. 43: 2861-2867].

All the components of the imaging device can be integrated into a single structure, such as an ergonomically designed closed structure with a handle, allowing it to be comfortably held with one or both hands. The device can also be provided without any handle. The device can be lightweight, portable and can allow real-time digital images (e.g., still and / or video images) of any target surface (e.g., the skin and / or the oral cavity, which is also accessible ) using white light, fluorescence and / or reflectance imaging modes. The device can be scanned across the body surface to obtain images by holding it at varying distances from the surface, and it can be used in an illuminated room / environment to obtain a white light reflectance / fluorescence image. The device can be used in a dark or dark room / environment to optimize the fluorescence signals of the tissue and minimize the background signals of the room lights. The device can be used for direct (e.g., naked eye) or indirect (e.g., through the digital imaging device display) display of wounds and surrounding normal tissues.

The device can also be incorporated by not being manual or portable, for example, by being fixed to a mounting mechanism (e.g., a tripod or stand) for use as a relatively stationary optical image device for white light images, fluorescence and reflectance of objects, materials and surfaces (e.g., a body). This may allow the device to be used on a desk or table or for "assembly line" images of objects, materials and surfaces. In some embodiments, the mounting mechanism may be mobile.

Other features of this device may include the ability to record digital video and image, possibly with audio, documentation methods (e.g., with image storage and analysis software) and wired or wireless data transmission for telemedicine needs / E-health remote. For example, e) and f) of Figure 2 show an embodiment of the device in which the image acquisition device is a mobile communication device such as a mobile phone. The mobile phone used in this example is a Samsung Model A-900, which is equipped with a 1.3 megapixel digital camera. The phone is installed in the support frame for convenient images. e) shows the use of the device to obtain images of a sheet of paper with fluorescent ink that shows the word "Wound", f) shows images of the fingers stained with fluorescent ink and detection of common skin bacteria P. Acnes. Images from the mobile phone can be sent wirelessly to another mobile phone or wirelessly (e.g., through Bluetooth connectivity) to a personal computer for image storage and analysis. This demonstrates the ability of the device to perform real-time manual fluorescence imaging and wireless transmission to a remote site / person as part of a telemedicine / E-health wound care infrastructure.

In order to demonstrate the capabilities of the imaging device in wound care and other relevant applications, several feasibility experiments were carried out using the particular example described. It should be noted that during all fluorescence imaging experiments, the Sony camera (Sony Cybershot DSC-T200 digital camera, Sony Corporation, North America) was set up so that the images were captured without flash, and with the "Macro" image mode set. The images were captured at 8 megapixels. The flash was used to capture white light reflectance images. All images were stored on the xD memory card for later transfer to a personal computer for long-term storage and image analysis.

All white light and fluorescence reflectance images / films captured with the device were imported into Adobe Photoshop for image analysis. However, the image analysis software was designed using MatLab ™ (Mathworks) to allow a variety of image-based spectral algorithms (eg, fluorescence ratios from red to green, etc.) to be used to extract data of relevant images (e.g., spatial and spectral data) for quantitative detection / diagnostic value. Image post-processing also included the mathematical manipulation of images.

Imaging of bacteriological samples

The imaging device may be useful for imaging and / or supervision in clinical microbiology laboratories. The device can be used to obtain quantitative images of bacterial colonies and quantify colony growth in common microbiological assays. Fluorescence images of bacterial colonies can be used to determine growth kinetics. The software can be used to provide an automatic count of bacterial colonies.

To demonstrate the usefulness of the device in a bacteriology / culture laboratory, live bacterial cultures were cultured in sheep blood agar plates. Bacterial species include streptococcus pyogenes, serratia marcescens, staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas aeruginosa (American Type Culture Collection, ATCC). These were grown and maintained under conventional incubation conditions at 37 ° C and used for experimentation during the "exponential growth phase". Once colonies were detected in the plates (approximately 24 h after inoculation), the device was used to obtain images of agar plates containing individual bacterial species in a dark room. Using violet / blue excitation light (approximately 405 nm), the device was used to obtain combined green and red autofluorescence images (approximately 490-550 nm and approximately 610 640 nm emission) and only red autofluorescence (approximately 635 / - 10 nm, the maximum emission wavelength for endogenous fluorescent porphyrins) of each agar plate. Fluorescence images of each bacterial species were taken over time to compare and monitor colony growth.

Reference is now made to Figure 3. a) shows the device used to obtain images of live bacterial cultures that grow on sheep blood agar plates to detect bacterial autofluorescence. b) shows the autofluorescence image emitted by pseudomonas aruginosa. The device can also be used to detect, quantify and / or monitor the growth of bacterial colonies over time using fluorescence, as demonstrated in c) with fluorescence images of autofluorescent staphylococcus aureus growth on a 24-hour agar plate after inoculation. Notice the presence of different individual bacterial colonies in the image below. Using violet / blue excitation light (e.g., 405 nm), the device was used to detect both combined green and red emission autofluorescence (e.g., 490-550 nm 610-640 nm) and only red ( eg, 635 / - 10 nm, the maximum emission wavelength for endogenous fluorescent porphyrins) of several live bacterial species, including streptococcus pyogenes, shown in d); serratia marcescens, shown in e); staphylococcus aureus, shown in f); staphylococcus epidermidis, shown in g); Escherichia coli, shown in h); and pseudomonas aeruginosa, shown in i). Note that autofluorescence images obtained by the bacterial colonies device can provide useful image contrast for simple longitudinal quantitative measurements of bacterial colonization and growth kinetics, as well as a means of potentially monitoring the response to the intervention. therapeutic, with antibiotics, photodynamic therapy (PDT), low level light therapy, hyperbaric oxygen therapy (HOT) or advanced wound care products, as examples.

The high spatial resolution of the camera detector combined with a significant signal image to bacterial autofluorescence noise with the device allowed the detection of very small colonies (e.g., <1 mm in diameter). The device provided a sensitive and portable means to obtain images of individual bacterial colonies that grow on conventional agar plates. This provided a means to quantify and monitor the growth kinetics of bacterial colonies, as seen in c), as well as potentially monitor the response to therapeutic intervention, with antibiotics or photodynamic therapy (PDT) as examples, over time using fluorescence. . Therefore, the device can serve as a useful tool in the microbiology laboratory.

Figure 3J shows an example of the use of the imaging device in a) conventional bacteriology laboratory practice, b) in this case, the fluorescence image of a Petri dish containing Staphylococcus aureus combined with analysis software Exclusive imaging allows bacterial colonies to be counted rapidly, and in this case the fluorescence image of the culture plate shows ~ 182 (+/- 3) colonies (bright bluish green spots) that grow on agar at 37 ° C ( excitation of approximately 405 nm, emission of approximately 500-550 nm (green), emission of approximately> 600 nm (red)).

In addition to providing detection of bacterial strains, the device can be used to differentiate the presence and / or location of different bacterial strains (e.g., Staphylococcus aureus or Pseudomonas aeguginosa), for example in wounds and surrounding tissues. This may be based on the different autofluorescence emission badges of different bacterial strains, including those of the emission wavelength bands at 490-550 nm and 610-640 nm when excited by violet / blue light, such as light around 405 nm. Other combinations of wavelengths can be used to distinguish between other species in the images. This information can be used to select the appropriate treatment, such as the antibiotic choice.

Such imaging of bacteriology samples may be applicable to wound care supervision.

Use in wound healing supervision

The device can be scanned above any wound (e.g., on the surface of the body) so that the excitation light can illuminate the area of the wound. Then, the wound can be inspected using the device, so that the operator can see the wound in real time, for example, through a viewfinder on the imaging device or through an external display device (e.g. , a vertical image, a television screen, a computer monitor, an LCD projector or a viewfinder helmet). It may also be possible to transmit the images obtained from the device in real time (e.g., through wireless communication) to a remote viewing site, for example for telemedicine purposes, or send the images directly to a printer or Computer memory storage. The image can be performed within the routine clinical evaluation of the patient with a wound.

Before imaging, fiduciary markers (eg, using an indelible fluorescent ink pen) can be placed on the surface of the skin near the edges or perimeter of the wound. For example, four points, each of a different fluorescent ink color from separate indelible fluorescent ink pens, which can be provided as a kit to the clinical operator, can be placed near the margin or boundary of the wound on the surface of normal skin. . The device can obtain images of these colors using the excitation light and a multispectral band filter that matches the emission wavelength of the four ink dots. An image analysis can then be performed, by joint registration of fiduciary markers for alignment between images. Therefore, the user may not have to align the imaging device between different image sessions. This technique can facilitate obtaining longitudinal images (i.e., over time) of the wounds, and the clinical operator, therefore, can obtain images of a wound over time without aligning the device. Image formation during each image acquisition.

In addition, to help calibrate the intensity of fluorescence images, a conventional disposable single fluorescence "strip" can be placed in the field of vision during imaging of the wound (eg, using a soft adhesive that stick the strip to the skin temporarily). The strip may be impregnated with one or several different fluorescent dyes of varying concentrations that may produce predetermined and calibrated fluorescence intensities when illuminated by the excitation light source, which may have wavelengths or bands of emission wavelengths of single (e.g., 405 nm) or multiple fluorescence for image intensity calibration. The disposable strip can also have the four dots as described above (e.g., each of different diameters or sizes and each of a different fluorescent ink color with a single black dot placed next to it) of ink pens indelible fluorescent separated. With the strip placed near the margin or boundary of the wound on the normal surface of the skin, the device can be used to take pictures of white light and fluorescence. The strip can offer a convenient way to take multiple images over time of a particular wound and then align the images by analyzing images. Likewise, the fluorescent "intensity calibration" strip may also contain an additional linear measuring device, such as a fixed length ruler to aid in spatial distance measurements of wounds. Such a strip can be an example of a calibration target that can be used with the device to help calibrate or measure image parameters (e.g., wound size, fluorescence intensity, etc.), and another can be used similar calibration target.

It may be desirable to increase the consistency of the image results and reproduce the distance between the device and the wound surface, since the intensity of tissue fluorescence may vary slightly if the distance changes during multiple imaging sessions. Therefore, in one embodiment, the device may have two light sources, such as low power laser beams, which can be used to triangulate individual rays on the surface of the skin to determine a fixed or variable distance between the device and the wound surface. This can be done using a simple geometric arrangement between the laser light sources, and can allow the clinical operator to easily visualize the laser points on the skin surface and adjust the distance of the device to the wound during several imaging sessions. Other methods for maintaining a constant distance may include the use of ultrasound, or the use of a physical measurement, such as a ruler.

Use in white light imaging

The device can be used to take white light images of the total wound with normal surrounding normal tissues using a measuring device (eg, a ruler) placed within the field of view of the image. This may allow a visual evaluation of the wound and the calculation / determination of quantitative parameters such as the area, circumference, diameter and topographic profile of the wound. Wound healing can be assessed by planimetric measurements of the wound area at multiple times (e.g., in clinical visits) until wound healing. The duration of wound healing can be compared with the expected healing time calculated by means of multiple moment measurements of wound radius reduction using the equation R = V (A) / n (R, radius; A, planimetric wound area; constant n 3.14). This quantitative information about the wound can be used to track and monitor changes in the appearance of the wound over time, in order to assess and determine the degree of wound healing caused by natural means or by any therapeutic intervention. These data can be stored electronically in the patient's medical record for future reference. The operator can make white light images during the initial clinical evaluation of the patient.

Use in autofluorescence imaging

The device may be designed to detect all or a majority of autofluorescence (AF). For example, using a multispectral band filter, the device can obtain images of tissue autofluorescence emanating from the following tissue biomolecules, as well as the optical absorption associated with blood, for example, under 405 nm excitation: collagen (types I, II, III, IV, V and others) that have a green color, elastin that has a yellow-orange-yellow color, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), that emit a blue-green autofluorescence signal bacteria / microorganisms, most of which appear to have a broad emission of autofluorescence (e.g., green and red).

Image analysis may include the calculation of an AF ratio from red to green in the image. Intensity calculations can be obtained from regions of interest in the wound images. Pseudocolor images can be mapped on the white light images of the wound.

Examples in wound healing

Reference is now made to Figure 4. The device was tested on a model of wounds contaminated with bacteria. For this, pork, with skin, was bought from a butcher. To simulate the wounds, a scalpel was used to make incisions, which vary in size from 1.5 cm2 to 4 cm2 in the skin and deep enough to see the muscular layer. The device was used to obtain images of some meat samples without the addition of bacteria to the simulated wounds. For this, the meat sample was left at room temperature for 24 h for the bacteria in the meat to grow, and then images were made with the device using both white light reflectance and autofluorescence, for comparison.

To test the device's ability to detect connective tissues and several common bacteria present in typical wounds, a sample of pig meat with simulated wounds was prepared by applying six bacterial species to each of six small 1.5 wound incision sites. cm2 on the surface of the skin: streptococcus pyogenes, serratia marcescens, staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas aeruginosa. An additional small incision was made in the skin of the meat, in which no bacteria were added, to serve as a control. However, it was expected that bacteria from the other six incision sites could contaminate this site in time. The device was used to obtain images of the bacteria-laden meat sample using white light reflectance emission and violet / blue light-induced tissue autofluorescence, using a dual emission band filter (450-505 nm and 590-650 nm) and a single band emission filter (635 / - 10 nm) on the left and a single band filter over three days, at 24-hour intervals, during which the meat sample was kept at 37 ° C Imaging was also performed in an extruded polystyrene container in which the meat sample was stored during the three days.

Figure 4 shows the results of the device used for non-invasive detection of bacterial autofluorescence in a simulated animal wound model. In conventional white light images, the bacteria were hidden inside the wound site, as shown in a) and expanded in b). However, under the violet / blue excitation light, the device was able to allow the identification of the presence of bacteria at the wound site based on the dramatic increase in the red fluorescence of bacterial porphyrins against a background of green fluorescence bright connective tissue (e.g., collagen and elastins) as seen in c) and enlarged in d). The comparison of b) and d) shows a dramatic increase in the red fluorescence of the bacterial porphyrins against a bright green fluorescence background of the connective tissue (e.g., collagen and elastins). It was observed that with autofluorescence, bacterial colonies were also detected on the surface of the skin based on their green fluorescence emission, which caused the individual colonies to appear as green spots on the skin. These were not seen under a white light test. Fluorescence imaging of connective tissues helped determine wound margins as seen in e) and f), and some areas of the skin (marked with in c) appeared more red fluorescent than other areas, possibly indicates a subcutaneous infection by bacteria that produce porphyrin, e) and f) also show the device that detects fluorescent red bacteria in the surgical wound, which are hidden under images of white light.

The device mapped the biodistribution of bacteria at the site of the wound and in the surrounding skin and, therefore, can help target specific areas of tissues that require sampling or biopsy for microbiological tests. In addition, the use of the imaging device may allow monitoring of the response of bacteria-infected tissues to a variety of medical treatments, including the use of antibiotics and other therapies, such as photodynamic therapy (PDT), hyperbaric oxygen therapy ( HOT), low level light therapy, or anti-matrix metalloproteinase (MMP). The device can be useful for visualizing bacterial biodistribution on the surface, as well as within the depth of the wound tissue, and also for surrounding normal tissues. The device can be useful to indicate the spatial distribution of an infection.

Use of the device with contrast agents in wound monitoring

The device can be used with exogenous contrast agents, for example, the prodrug aminolevulinic acid (ALA) in a low dose. The ALA can be administered topically to the wound and the image can be done 1-3 hours later to improve the red fluorescence of the wound bacteria.

The prodrug aminolevulinic acid (ALA) induces the formation of porphyrin in almost all living cells. Many species of bacteria exposed to ALA can induce protoporphyrin IX (PpIX) fluorescence. The use of ultra-low dose ALA can induce the formation of PpIX in the bacteria and, therefore, can increase the emission of red fluorescence, which can enhance the contrast of red to green fluorescence of the bacteria with the device. ALA is not fluorescent by itself, but PpIX is fluorescent at approximately 630 nm, 680 and 7 10 nm, with the emission of 630 nm being the strongest. The imaging device can be used to obtain images of the green and red fluorescence of the wound and surrounding tissues. The time required to obtain a significant / appreciable increase in red fluorescence (e.g., peak at 630 nm) using the imaging device after application of ALA (~ 20 pg / ml) at the intervals of the wound of 10-30 minutes, but this time can be optimized, and it also depends on the dose of ALA that can also be optimized.

Therefore, a clinical operator can premix the ALA, which is usually commercially provided in lyophilized form with physiological saline solution or other commercially available cream / ointment / hydrogel / dressing, etc., in a given dose and administer the agent via Topical by spraying, pouring or carefully applying the agent to the wound area before imaging. Approximately 10-30 minutes later, although this time may vary, the fluorescence image can be performed in a dimly lit or dark room. Bacteria hidden under white light and, perhaps, little autofluorescent, may appear as bright red fluorescent areas in the wound and around it. Fluorescence images can be used to direct sampling, biopsy and / or fine needle needle aspirates of the wound for bacterial culture based on the single bacterial fluorescence signal, and this can be done at different depths, for wounds Superficial and deep

The device can also be used together with exogenous "prodrugs" agents, including, but not limited to, ALA, which is approved by the FDA for clinical therapeutic indications, to increase endogenous porphyrin production in bacteria / microorganisms and therefore increase the intensity of the unique "porphyrin" fluorescence signals emanating from these bacteria to improve the detection sensitivity and specificity of the device. Accordingly, the device can be used to conveniently visualize photosensitizer-induced fluorescence (e.g., PpIX) in bacteria, which grow in culture or in patient wounds for subsequent sampling / biopsy or guided training-guided treatment. of images, for example, using photodynamic therapy (PDT) or hyperbaric oxygen therapy (HOT). The device when used, for example, with commercially available consumable fluorescence contrast agents has the ability to increase the signal to the bottom for sensitive detection of bacteria, in wounds and around them. It should be noted that ALA is commercially available.

In one example, the device was used to obtain images of live bacterial cultures (staphylococcus aureus, cultured on agar plates for 24 h before imaging) using blue / violet excitation light. After 30 minutes of incubation of staphylococcus aureus -20 pg / ml of ALA at 37 ° C, a significant increase in the red fluorescence of the bacteria was detected, compared to those colonies that did not receive ALA. Accordingly, the device can exploit the use of contrast agent strategies to increase the signal to the bottom for the sensitive detection of bacteria, for example, in wounds. The time required for ALA to increase the PpIX fluorescence of bacteria in culture to significant levels was approximately 0.5 h, suggesting that this approach may be clinically practical. Tests on simulated meat samples contaminated with bacteria revealed similar results to those obtained from bacterial cultures. Topical application of 0.2 pg / ml of ALA by spraying on wounds in pig skin produced a dramatic increase in the fluorescence contrast of bacterial porphyrin red approximately 2 h after administration of ALA. This demonstrates that the device can allow the detection of bacterial contamination with fluorescence images at the wound sites and any other part of the skin surface, which was previously hidden under white light images.

Use with activated imaging agents targeting exogenous molecular groups

The availability of commercially available molecular bacteriological fluorescence detection and viability kits may offer another use for the device in wound care. Such kits can be used to quickly distinguish live and dead bacteria quantitatively, even in a mixed population that contains a range of bacterial types. Conventional direct count tests of bacterial viability are usually based on metabolic characteristics or membrane integrity. However, methods that depend on metabolic characteristics often only work for a limited subset of bacterial groups, and methods for assessing the integrity of the bacterial membrane generally have high levels of background fluorescence. Both types of determinations also experience being very sensitive to growth and staining conditions.

Suitable exogenous optical molecular targeting probes can be prepared using commercially available fluorescence labeling kits, such as Alexa Fluor active esters and kits (e.g., Zenon antibody marking kits and EnzChek protease assay kits). , Invitrogen) to label proteins, monoclonal antibodies, nucleic acids and oligonucleotides (Invitrogen). For example, these fluorescent dye bioconjugates cover the following wavelength ranges: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546 , Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750, in which the number indicated refers to the excitation wavelength of the dye. These kits can offer well differentiated fluorescence emission spectra, which provide many options for multicolored fluorescence detection and fluorescence resonance energy transfer, depending on the appropriate selection of fluorescence emission filters with the formation device. images. Fluorescence dyes offer high absorbance at maximum output wavelengths of common excitation sources, are bright and unusually photostable fluorescents of their bioconjugates, and offer good water solubility of reactive dyes to facilitate conjugation in the room. Clinical examination and resistance of the conjugates to precipitation and aggregation. The fluorescence spectra of the dyes are insensitive to pH over a wide range, which makes them particularly useful for obtaining wound images, since the pH of the wounds may vary. In addition, there are other commercial or non-commercial fluorescent agents that may be appropriate for obtaining biological images of the wounds and may be combined with the described device, including fluorescent blood accumulation agents and various probes activated by enzymes or wound proteases from VisEn Medical (Boston, Mass., USA), for example.

These directed fluorescent bioconjugates can be prepared using said marking kits before the clinical examination of the wound using the fluorescence mode imaging device, and can be stored in airtight containers to prevent photobleaching. Such fluorescence bioconjugates can be prepared in solution at a known and appropriate concentration before the fluorescence image of the wound using the device, and then administered / applied directly to the wound and surrounding normal tissues either topically (e.g. ., by aerosol / spray, washing techniques), or administered orally in a beverage or according to an example not covered by the method systemically claimed by intravenous injection. Such dyes may target specific biological components based on the target moiety, and may include: bacteria, fungi, yeasts, spores, viruses, microbes, parasites, exudates, pH, blood vessels, reduced nicotinamide adenine dinucleotide (NADH), adenine falvina dinucleotide (FAD), microorganisms, specific types of connective tissues (e.g., collagens, elastin), tissue components, vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), epithelial growth factor, membrane antigen of epithelial cells (ECMA), hypoxia-inducible factor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin, fibroblast growth factor, transforming growth factors (TGF), fibroblast activation protein ( FAP), enzymes (e.g., caspases, matrix metalloproteinases (MMPs), etc.), tissue inhibitors of metalloproteinases (e.g., TIMPs), nitric oxide synthase (NOS), NOS inducible and endothelial, lysosomes in cells, macrophages, neutrophils, lymphocytes, hepatocyte growth factor (HGF), anti-neuropeptides, neutral endopeptidases (NEP), granulocyte and macrophage colony stimulating factor (GM-CSF), neutrophil elastases , cathepsins, arginase, fibroblasts, endothelial cells and keratinocytes, keratinocyte growth factor (KGF), macrophage inflammatory protein-2 (MIP-2), macrophage inflammatory protein-2 (MIP-2) and chemoattractant protein-1 protein macrophage (MCP-1), polymorphonuclear neutrophils (PMN) and macrophages, myofibroblasts, interleukin-1 (iL-1) and tumor necrosis factor (TNF), nitric oxide (NO) (Calbiochem kit, model DAF-2 DA) , c-myc, beta-catenin, endothelial progenitor cells (EPCs) of the bone marrow. Exogenous optical agents may include, among others, any of the following: activated molecular beacons (e.g., directed), nanoparticles having fluorescent agents (e.g., surface marked and / or containing or carrying agents fluorescent), and dispersion or absorbent nanoparticles (e.g., gold, silver).

The LIVE / DEAD BacLight ™ bacterial viability kits assay (from Invitrogen, Molecular Probes) uses mixtures of SYTO® 9 green fluorescent nucleic acid staining and red fluorescent nucleic acid staining, propidium iodide, although these fluorescent dyes can be exchanged with other existing or emerging fluorescent agents. These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, SYTO 9 staining marks bacteria with intact and damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, competing with SYTO 9 staining for nucleic acid binding sites when both dyes are present. When Mixing in the recommended proportions, the staining of SYTO 9 and the propidium iodide produce a fluorescent green staining of bacteria with intact cell membranes and a fluorescent red staining of bacteria with damaged membranes. Therefore, live bacteria with intact membranes emit green fluorescence, while dead bacteria with damaged membranes emit red fluorescence. The background remains virtually non-fluorescent. Consequently, the ratio of green to red fluorescence intensities can provide a quantitative index of bacterial viability.

Live and dead bacteria can be seen separately or simultaneously by the imaging device with suitable optical filter assemblies. In addition, similar fluorescence test kits are available for the identification of bacteria with the Gram sign (ie positive / negative), which is a useful parameter in wound treatment planning, and can be used together with the device Image formation Such fluorescence agents are general and applicable to most types of bacteria, and can be used to determine bacterial viability and / or the Gram sign directly in / inside the wound or in culture samples derived from smears or ex vivo tissue biopsies obtained from the wound site (e.g., superficially or in depth) for quantitative evaluation in real time using the imaging device. Such fluorescence fluorescent agents can be prepared in solution in advance at a known and appropriate concentration before the fluorescence image of the wound using the device, and then administered / applied directly to the wound and surrounding normal tissues, either via topical (eg, by aerosol / spray, washing techniques), or perhaps systemically via intravenous injection. Then, the image can be taken accordingly after a defined time for the agents to react with the targets. A washing of unmarked agents may be required before taking pictures with the device. For this, physiological saline solution can be used. The fluorescent agent bound to the target can remain in the wound and surrounding tissues to obtain fluorescence images.

Therefore, when used with fluorescent indicator systems, the imaging device can provide a relatively rapid means of assessing bacterial viability after exposure to antimicrobial agents. The ability to repeatedly measure the same patients or animals can reduce the variability within treatment experiments and allow equal or greater confidence in determining the effectiveness of the treatment. This non-invasive and portable imaging technology can reduce the amount of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

A number of commercially available organic fluorophores have properties that depend on the concentration of hydrogen ions, making them useful as probes for measuring pH, and usually have UV / visible absorption properties sensitive to pH. Most commercially available pH-sensitive fluorescent dyes used in intracellular studies provide a reduced fluorescent signal in acidic media or, alternatively, the dye pKa is outside the critical intracellular pH window of between 5 and 8 pH units. . However, other pH sensitive fluorescent agents respond by increasing their fluorescence intensities. For example, Invitrogen / Molecular Probes offer a variety of fluorescent pH indicators, their conjugates and other reagents for pH measurements in biological systems. Among these are several probes with unique optical response and specialized location characteristics: for example, excitable visible light SNARF pH indicators allow researchers to determine intracellular pH in the physiological range using dual emission or dual excitation radiometry techniques , thus providing useful tools for confocal laser scanning microscopy and flow cytometry. LysoSensor probes, as well as indicators based on the Oregon Green fluorophore, can be used to estimate the pH in the acidic organelles of a cell. There are also fluorescent pH indicators coupled to dextrans that can be used. After loading in the cells, the indicator dextrans may be well retained, may not bind to cellular proteins and may have a reduced tendency to compartmentalize. Again, said fluorescent agents can be prepared in solution in advance at a known and appropriate concentration before the fluorescence image of the wound using the device, and then administered / applied directly to the wound and surrounding normal tissues either topically. (e.g., by aerosol / spray, washing techniques), systemically or for example, by intravenous or oral injection.

Examples

Reference is now made to Figure 24. As an example, the imaging device can be used clinically to determine the healing status of a chronic wound and the success of wound debridement. For example, the figure shows a typical foot ulcer in a person with diabetes, with (i) the unhealed edge (i.e. callus) containing ulcerogenic cells with molecular markers indicative of healing impairment and (ii) phenotypically normal cells but with physiologically altered cells, which can be stimulated to heal. Despite the appearance of a wound after debridement, it may not be healing and it may be necessary to evaluate the presence of specific molecular markers of inhibition and / or hyperkeratotic tissue (e.g., c-myc and p-catenin). Using the imaging device in combination with fluorescently labeled exogenous molecular probes against such molecular targets, the physician can determine the in situ expression of molecular biomarkers. With the device, once a wound was de-fused, fluorescence images of the wound area and image analysis can allow the orientation of the biopsy for subsequent immunohistochemistry and this can determine if the degree of debridement was sufficient. If the degree of debridement was insufficient, as shown in the lower left diagram, cells positive for c-myc (which has a green color) and nuclear p-catenin (which has a purple color) can be found depending on their fluorescence , which indicates the presence of ulcerogenic cells, which can prevent the wound from healing properly and indicate that additional debridement is necessary. The lack of scarring can also be demarcated by a thicker epidermis, a thicker cornified layer and the presence of nuclei in the cornified layer. If the debridement was satisfactory, as in the lower right lower diagram, no staining for c-myc or p-catenin can be found, indicating an absence of ulcerogenic cells and a successful debridement. These inhibition markers may be useful, but the goal is the actual healing defined by the appearance of a new epithelium, the decrease in the area of the wound and the absence of drainage. This information can be collected using the fluorescence imaging device and can be stored electronically in the patient's medical record, which can provide an objective analysis along with pathology and microbiology reports. By comparing the expected healing time with the actual healing time (i.e., the progress of healing) using the imaging device, adaptive treatment strategies can be implemented per patient.

Figure 24B shows an example of the use of the device to obtain images of the healing of a pressure ulcer. a) The white light image taken with the right foot device of a diabetic patient with a pressure ulcer is shown. b) The corresponding fluorescence image shows the bright red fluorescence of the bacteria (bacteriology results confirmed the presence of strong growth of Staphylococcus aureus) that are invisible under the conventional white light test (yellow arrows). Observe the strong growth of the Staphylococcus aureus bacteria around the periphery of the wound that does not heal (long yellow arrow). cd) shows the separate red-green-blue (unmixed) spectral images of the unprocessed fluorescence image in b), which are used to produce spectrally encoded image maps of green fluorescence intensities (e.g., collagen ) and red (e.g., bacteria) calculated using mathematical algorithms and displayed in false color with color scale. fg) shows examples of image processing methods used that improve the contrast of the endogenous bacterial autofluorescence signal by calculating the intensity ratio of red / green fluorescence to reveal the presence and biodistribution of bacteria (reddish-yellow) in the wound open and around it. These data illustrate the ability to use custom or commercially available image analysis software to mathematically analyze the fluorescence images obtained by the device and display them in a meaningful way for clinical use, and this can be done in real time. (1 cm scale bar).

Figure 24C shows an example of the use of the device to obtain images of a chronic wound without scarring. a) the white light image taken with the left breast device of a patient with Pyoderma gangrenosum shows a chronic wound that does not heal (blue arrow) and a healed wound (red arrow). Bacteria generally cannot be visualized by conventional visualization of white light used in the conventional clinical examination of wounds. b) the corresponding fluorescence image of the same wounds is shown (in this example, using excitation at 405 nm, emission of 500-550 nm (green), emission of> 600 nm (red)). Note that the unhealed wound has a dark color under fluorescence (mainly due to the absorption of light by excitation and fluorescence emission light from the blood), while bacteria appear as red dotted spots bright on the wound healed (red arrow). Under fluorescence, the normal surrounding skin has a cyan green color due to the fluorescence of the endogenous collagen (405 nm excitation). In contrast, the uncured wound (blue arrow) appears to have a very bright red fluorescence band around the edge of the wound, confirmed with smear cultures (bacteriology) that contains a large growth of Staphylococcus aureus (with few Gram positive bacilli and few Gram positive cocci , confirmed by microscopy). c) white light image of the wound healed in a, b) and d) corresponding fluorescence image showing bright red fluorescence of bacteria (pink arrows), which are hidden under white light. e) white light and f) corresponding fluorescence images of the uncured breast wound. Note that bacteria (Staphylococcus aureus) appear to be located mainly around the edge / boundary of the wound (yellow arrow), while there are fewer bacteria located in the wound (X), determined by the biodistribution of bacteria directly visualized by fluorescence images, but invisible under white light (black arrow, e). (Scale bar in cm).

Figure 24D further illustrates the imaging of a chronic wound without scarring using an example of the imaging device. a) White light image taken with the left breast device of a patient with Pyoderma gangrenosum , which shows a chronic wound that does not heal (blue arrow) and a healed wound (blue arrow). Bacteria cannot be visualized by conventional visualization of white light used in the clinical examination of wounds. b) corresponding fluorescence image of the same wounds (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)). While the nipple appears to be normal blank without obvious contamination of bacteria, the fluorescence image shows the presence of bacteria emanating from the nipple ducts. Nipple smears showed that the bacteria were Staphylococcus epidermidis (occasional growth found in the culture). (Scale bar in cm).

Figure 24E shows a central area and the edge of a chronic wound without scarring visualized with the imaging device. a) white light image taken with the left breast device of a patient with Pyoderma gangrenosum, which shows the central area and the edge of a chronic wound that does not heal. a) white light and b) corresponding fluorescence images of the wound in the uncured sinus (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)). Note that bacteria (Staphylococcus aureus; shown by bacterial sampling) appear to be located mainly around the edge / boundary of the wound, while there are fewer bacteria located in the wound (X), determined by the biodistribution of bacteria directly visualized by fluorescence images, but invisible under white light. (Scale bar in cm).

Figure 24F shows additional images of a chronic wound without scarring using the imaging device. a) white light image taken with the left breast device of a patient with Pyoderma gangrenosum , which shows a chronic wound that does not heal. Bacteria cannot be visualized by conventional visualization of white light used in the clinical examination of wounds. b) corresponding fluorescence image of the same wound (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)). Fluorescence images show the presence of bacteria around the edge / margin of the wound before cleaning (b) and after cleaning (c). In this example, cleaning involved the use of conventional gauze and phosphate buffered saline to clean the surface of the wound (inside and outside) for 5 minutes. After cleaning, the red fluorescence of the bacteria decreases considerably, indicating that some of the red fluorescent bacteria may reside beneath the surface of the tissue around the edge of the wound. Small amounts of bacteria (red fluorescent) remained in the center of the wound after cleaning. This illustrates the use of the imaging device to monitor the effects of wound cleansing in real time. As an additional example, d) shows a white light image of a chronic wound that does not heal in the same patient located in the left calf. e) shows the corresponding fluorescence images before cleaning (e) and after cleaning (f). Sampling of the central area of the wound revealed the occasional growth of Staphylococcus aureus, with strong growth of Staphylococcus aureus on the edge (yellow arrow). Cleaning resulted in a reduction of fluorescent bacteria ( Staphylococcus aureus ) on the surface of the wound as determined using the optical manual imaging device. The use of the imaging device resulted in real-time detection of hidden white light bacteria and this allowed an alteration in the way the patient was treated, so that, after fluorescence imaging, the wounds and the surroundings (contaminated bacteria) were thoroughly cleaned again or cleaned for the first time due to de novo bacteria detection. Also, keep in mind the use of a "strip" of calibration-measuring disposable adhesive to aid in the imaging approach and this "strip" can adhere to any part of the body surface (eg, near of a wound) to allow spatial measurements of the wound. The calibration strip can also be clearly fluorescent and can be used to add patient-specific information to the images, including the use of multiple exogenous fluorescent dyes for barcode purposes, whose information can be integrated directly into the fluorescence images of the wounds. (Scale bar in cm).

Figure 24G illustrates the use of the imaging device to monitor wound healing over time. The imaging device is used to track changes in the healing status and bacterial biodistribution (e.g., contamination) of a chronic wound that does not heal from the left breast of a patient with Pyoderma gangrenosum. Over six weeks, white light images (am) and corresponding fluorescence images of the healed wound (bn) and the chronic non-healing wound (co) are shown. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)), taken using the imaging device in white light and fluorescence modes. In bn), the presence of small bacterial colonies of bright red fluorescence (yellow arrows) is detected and their location changes over time in the healed wound. Bacterial smears confirmed that no bacteria were detected under the microscope and no bacterial growth was observed in the culture. In co), on the contrary, the uncured wound has a very bright red fluorescence band around the edge of the wound, confirmed with smear cultures (bacteriology) that contain a large growth of Staphylococcus aureus (with few Gram positive bacilli and few Gram positive cocci , confirmed by microscopy), which changes in biodistribution over time (i.e., co). These data demonstrate that the imaging device can provide biological and molecular information in real time, as well as be used to monitor morphological and molecular changes in wounds over time.

Figure 24H shows another example of the use of the device to monitor the condition of the wound over time. The imaging device is used to track changes in the healing status and bacterial biodistribution (e.g., contamination) of a left calf wound of a 21-year-old patient with Pyoderma gangrenosum. The white light images (ai) and the corresponding fluorescence images of (bj) a wound treated with hyperbaric oxygen therapy (HOT) are shown over six weeks. (Fluorescence parameters: 405 nm excitation, 500-550 nm emission (green), emission> 600 nm (red)), ai) white light images reveal different macroscopic changes in the wound as it heals, which It is indicated by the reduction in size over time (e.g., closing) from week 1 (diameter ~ 2 cm long) to week 6 (shaft diameter ~ 0.75 cm long). In bj), the real-time fluorescence image of endogenous bacterial fluorescence (autofluorescence) in and around the wound can be traced over time and correlated with white light images and wound closure measurements (ai ). b) shows a clear green band of fluorescence at the immediate limit of the wound (yellow arrow; it is shown that it is contaminated with abundant growth of Staphylococcus aureus), and this band changes over time as the wound heals. Red fluorescence bacteria are also seen farther from the wound (orange arrow) and their biodistribution changes over time (bj). The limits of wound tissue to perilesional to normal can be clearly seen by fluorescence in image j). Connective tissue (in this example, collagens) in normal skin appears as a pale green fluorescence (j) and remodeling of connective tissue during wound healing can be monitored over time, during various wound treatments. which include, as is the case, hyperbaric oxygen therapy of chronic wounds.

Figure 24I illustrates the use of the imaging device to detect bacterial smears during routine wound evaluation in the clinic. In fluorescence imaging, the sample can be directed or select specific areas of bacterial contamination / infection using the real-time fluorescence imaging guide. This can decrease the potential for contamination of uninfected tissues by reducing the spread of bacteria during routine sampling procedures, which can be a problem in conventional wound sampling methods. The smear results of this sample were determined to be from Staphylococcus aureus (with few Gram positive bacilli and few Gram positive cocci , confirmed by microscopy).

Figure 24J shows an example of the co-registration of a) white light and b) corresponding fluorescence images made with the imaging device in a patient with uncured foot ulcers associated with diabetes. Using a non-contact temperature measuring probe (box in a) with a cross-laser viewfinder, direct temperature measurements were made on normal skin (yellow "3 and 4") and on foot ulcers (yellow "1 and 2" ) (infected with Pseudomonas aeruginosa, as confirmed by bacteriological culture), indicating the ability to add temperature-based information to the wound assessment during the clinical examination. Infected wounds have elevated temperatures, as seen in the average of 34.45 ° C in infected wounds compared to 30.75 ° C on the normal skin surface, and these data illustrate the possibility of multimodal measurements that They include white light, fluorescence and thermal information for wound health assessment / infectious evaluation in real time. Note that both wounds that do not heal on the right foot of this patient contained a large growth of Pseudomonas aeruginosa (in addition to the Gram positive cocci and the Gram negative bacilli), which in this example appear as bright green fluorescent areas in the wound (b).

Figure 24K shows an example of the use of the imaging device to monitor a pressure ulcer. a) the white light image taken with the imaging device of the right foot of a Caucasian diabetic patient with a pressure ulcer is shown. b) the corresponding fluorescence image shows the bright red fluorescence of the bacteria (bacteriology results confirmed the presence of strong growth of Staphylococcus aureus) that are invisible under the conventional white light test (yellow arrows). Dead skin appears as a green color of white / pale light (white arrows). Observe the strong growth of the Staphylococcus aureus bacteria around the periphery of open wounds that do not heal (yellow arrows). c) shows the fluorescence image of a topical application silver antimicrobial dressing. The imaging device can be used to detect the endogenous fluorescence signal of advanced wound care products (e.g., hydrogels, wound dressings, etc.) or the fluorescence signals of such products that have been prepared. with a fluorescent dye with an emission wavelength within the detection sensitivity of the imaging detector in the device. The device can be used for administration / application guided by images of advanced products for the treatment of wound care and to subsequently monitor its distribution and purification over time.

Figure 24L shows an example of the use of the device to monitor a pressure ulcer. a) white light image taken with the device of the right foot of a caucasian diabetic patient with a pressure ulcer. b) the corresponding fluorescence image shows the bright red fluorescent area of the bacteria (the bacteriological results confirmed the presence of intense growth of Staphylococcus aureus , SA ) at the edge of the wound and the bright green fluorescent bacteria (the bacteriological results confirmed the presence of intense growth of Pseudomonas aeruginosa, PA) that are invisible under the conventional white light test, c) the fluorescence spectroscopy taken from the wound revealed unique spectral differences between these two bacterial species: SA has an emission peak of characteristic red autofluorescence (approximately 630 nm), while PA lacks red fluorescence but has a strong green autofluorescence peak at around 480 nm.

Figure 24M shows an example of the use of the device to monitor a chronic wound that does not heal. a) the white light image taken with the chronic wound imaging device that does not heal in a 44-year-old black male patient with type II diabetes is shown. Bacteria cannot be visualized by the conventional white light visualization (ag) used in the conventional clinical examination of wounds, bh) corresponding fluorescence image of the same wounds (405 nm excitation, 500-550 nm emission (green) , emission of> 600 nm (red)). This patient presented multiple open wounds without scarring. Smear cultures taken from each area of the wound using the fluorescence imaging guide revealed abundant growths of Pseudomonas aruginosa (yellow arrow) that have a bright green fluorescent color, and Serratia marcescens (circles) that has a red fluorescent color. (Scale bar in cm).

Figure 24N is a schematic diagram illustrating an example of a use of "calibration" targets, which can be custom designed, multipurpose and / or disposable, for use during wound imaging with the device for forming images. The strip, which in this example is adhesive, may contain a combination of one or more of: spatial measurement tools (e.g., length scale), information barcode to integrate patient-specific medical information and gradients of concentration impregnated with fluorescent dyes for real time fluorescence image calibration. For the latter, multiple concentrations of various exogenous fluorescent dyes or other fluorescence agents (e.g., quantum dots) can be used for calibration of multiplexed fluorescence intensity, for example, when more than one exogenous probe labeled with fluorescence for tissue / cell / molecular molecular images of wounds in vivo.

Figure 24O shows an example of the use of an embodiment of the imaging device to monitor bacteria, for example, to monitor a treatment response, a) fluorescence microscopy image of a live / dead bacteria stain sold by Invitrogen Corp (i.e., product of BacLight), b) fluorescence microscopy image of a bacterial staining stain with Gram stain sold by Invitrogen Corp. By using the imaging device (c) with said products, the bacteria live (green) and dead (red) (e) can be distinguished in real time ex vivo (e.g., in the smear or tissue biopsy) followed by bacterial sampling of a wound, or other body surface, by example, in the sampling of the oral buccal cheek, as in d). This real-time Gram bacterial staining or evaluation based on live / dead images may be useful for real-time or relatively rapid bacteriology results that can be used for refining treatments, such as antibiotics or other disinfectant treatments, or for monitoring treatment response

Figure 24P shows an example of the use of the device used to obtain images of the infection of the toenail, a) white light and b) corresponding autofluorescence of the right toe of a subject demonstrating the improved contrast of the infection that it provides the fluorescence image compared to the white light display (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)).

Figure 24Q shows an example image using the device to monitor the response of meat infected by bacteria with a disinfectant (eg, hydrogen peroxide (Virox5TM)). a) A sample of swine tissue ex vivo was prepared in Petri dishes and contaminated with Staphylococcus aureus before topical administration of b) Virox5TM and fluorescence imaging (with a handheld device), c). Tissue breakdown begins to occur rapidly, caused by the disinfectant, while a change in the fluorescence characteristics of the bacteria is observed (e.g., the color of red fluorescence begins to change to an color of orange fluorescence, such as it can be seen in d), especially after gentle agitation of the sample and over time, here approximately 5 minutes of incubation with the Virox5TM solution. These data suggest the use of the device to monitor bacterial disinfection, for example, in clinical and non-clinical settings (excitation of 405 nm; emission of 490-550 nm and> 600 nm).

In addition to prodrugs that increase fluorescence, advances in medicine have allowed the widespread use of fluorescent biomarkers to diagnose diseases at the molecular level. Accurate measurement of the fluorescent biomarker signal in biological tissues can be a critical parameter for obtaining biomolecular information on disease progression and response to treatment, but historically it has posed a significant challenge. To date, this type of advanced molecular imaging has not been reported for wound care.

The device described herein can also be used in combination with fluorescent, light dispersants or exogenous light absorbing fluorescence contrast agents that can be used passively and / or directed at single and specific molecular targets in the wound for improve the detection and diagnosis of wound infection. These targets can be any biological and / or molecular component in the wound or normal surrounding tissues that have a known detection and / or diagnostic value (e.g., biomarkers of normal tissues and wounds). All exogenous agents can be administered in the wound topically and / or systemically, and can include, among others, any exogenous agent / drug (e.g., encapsulated liposomes, beads or other biocompatible vehicle agents) that can be coupled / conjugate with an appropriate fluorescent moiety / dispersers selected at appropriate wavelength (e.g., organic fluorescent dyes, quantum dots and other fluorescent semiconductor nanoparticles, colloidal metals (e.g., gold, silver, etc.)). Fluorescent agents / probes and / or light dispersers, and / or chromogenic agents / dyes (ie, absorption) can be prepared using conventional bioconjugate techniques to include moieties to target specific biomarkers. Such moieties may include monoclonal antibodies (e.g., whole and / or fragments), and other tissue specific moieties (including, among others, peptides, oligomers, aptamers, receptor binding molecules, enzyme inhibitors, toxins, etc. ). The device can also be used to obtain in situ images of expression controlled by an activatable promoter of light generating proteins in preclinical wound models. In addition, wound infections can also be detected using the imaging device and then treated with photothermal therapies, such as light-absorbing gold nanoparticles. conjugated with specific antibodies that specifically target bacteria.

Figure 24R shows an example of the use of the imaging device used to obtain images of dyes / probes / fluorescent agents in biological tissues, a) the white light image of a piece of meat (ex vivo) does not reveal the presence of a fluorescent dye, while in b) the device allows the precise detection of fluorescence and the monitoring of the biodistribution of the fluorescent dye. Although it is shown for ex vivo tissue , these capabilities can be transferred to in vivo applications that include, among others, images of the biodistribution of fluorescent photosensitizers in tissues for photodynamic therapy (PDT) of wounds, cancer, infection or other diseases. White light images can provide an anatomical context for fluorescence images. These capabilities can also be used to monitor the photobleaching of fluorescent agents (including photosensitizers), as well as for image-guided administration of multiple PDT treatments (405 nm excitation, 500-550 nm emission (green), emission of > 600 nm (red)). The device can provide monitoring of pharmacokinetics, biodistribution and / or photobleaching in PDT. Similarly, the device can be useful for monitoring low level light therapies.

The device can also be used with other molecular detection agents, such as "molecular beacons" or "smart probes," which produce fluorescence only in the presence of unique and specific biological targets (eg, enzymes associated with the health of the wound). Such probes may be useful for identifying specific or distinctive GRAM bacterial species, for example. For example, healing of skin wounds is a highly complex process that involves five overlapping phases (inflammation, granulation tissue formation, epithelization, matrix production and remodeling) associated with a series of migratory and remodeling events that are believed to require the action of matrix metalloproteinases (MMPs) and their inhibitors, TIMPs. In vivo analyzes of acute and chronic human wounds, as well as a variety of different wound healing models, have involved a functional role of MMPs and TIMPs during normal wound repair, while it is believed that the Deregulation of its activity contributes to the healing of wounds. Degradation of extracellular matrices is necessary to remove damaged tissue and provisional matrices and to allow vessel formation and reepithelialization. In contrast, in chronic wounds or without scarring, it is thought that overexpression of proteinases in their inactive form contributes to the underlying pathology and inhibits normal tissue repair processes. Molecular beacons are activatable fluorescent indicators that use the principle of fluorescence resonance energy transfer (FRET) to control fluorescence emission in response to specific biological stimuli. They generally comprise a disease-specific linker that brings an inhibitor dye closer to a fluorophore to silence its fluorescence. Following specific interactions between the linker and the target (eg, nucleic acid hybridization, protease specific peptide cleavage, phospholipase specific phospholipid cleavage), the inhibitor dye is removed from the neighborhood of the fluorophore to restore its fluorescence. These smart probes can offer several orders of magnitude sensitivity that direct the probes due to the high built-in degree of signal amplification from non-fluorescent to highly fluorescent. Depending on their specific linker-target interactions, they may also be able to interrogate specific molecular abnormalities in protein or gene expression levels. Because of these advantages, smart probes have recently been hailed as "a quantum leap" compared to traditional probes for early cancer detection. Such exogenous agents can be used, for example, for the relatively rapid, non-invasive, sensitive and specific optical detection of wound infections, to identify specific bacterial species / microorganisms present and microbial diagnosis in situ, to monitor the health status of the wound, and notify in real time about the effectiveness of treatment and care.

In addition, when used in combination with exogenous optical agents, the device can be used to identify patients who respond minimally to various established and experimental treatments, allowing rapid non-invasive or non-contact quantitative visual evaluation of the treatment response. for timely changes in therapy in order to optimize the clinical outcomes of the treatment.

In addition, real-time monitoring of antimicrobial effects in vitro and in animal model test systems using the imaging device can improve basic understanding of the action of antibiotics and facilitate unique studies of diseases in vivo.

Examples

Figure 5 shows an example of the device that is used for the non-invasive autofluorescent detection of collagen and varies the bacterial species on the skin surface of a sample of pork. Unlike the white light images, the autofluorescence images were able to detect the presence of several bacterial species 24 h after they were applied topically in small incisions made in the skin (i.e. streptococcus pyogenes, serratia marcescens, staphylococcus aureus , staphylococcus epidermidis, escherichia coli, and pseudomonas aeruginosa). a) shows white light images of the pork used for the test. Several bacterial species were applied to small incisions made on the skin on day 0, and they were marked as follows: 1) streptococcus pyogenes, 2) serratia marcescens, 3) staphylococcus aureus, 4) staphylococcus epidermidis , 5) escherichia coli , and 6) pseudomonas aeruginosa . The imaging device was used to detect collagen and bacterial autofluorescence over time. The fluorescence of the connective tissue was intense and was also easily detected. Some bacterial species (e.g., pseudomonas aeruginosa) produce a significant green autofluorescence (450-505 nm) that saturated the device chamber, b) shows the autofluorescence image on day 0, enlarged in c).

The device was also able to detect the spread of bacteria on the surface of the meat over time. d) shows an image on day 1, and f) shows an image on day 3, since the meat sample was kept at 37 ° C. A red fluorescence can be observed at some of the wound sites (5, 6) in c). As shown in d) and amplified in e), after 24 h, the device detects a dramatic increase in the bacterial autofluorescence of the wound site 5) escherichia coli and 6) pseudomonas aeruginosa, the latter producing a significant green autofluorescence and red c) and e) show the device that detects fluorescence using a dual band (450-505 nm green and 590-650 nm) on the left and a single band filter (635 / - 10 nm) on the right, from the surface of the wound. As shown in f), on day 3, the device detects the significant increase in bacterial autofluorescence (in green and red) of the other wound sites, as well as bacterial contamination (indicated by the arrow in f) in the extruded polystyrene container in which the meat sample was preserved. The device was also able to detect the spread of bacteria on the surface of the meat. This demonstrates the real-time detection of bacterial species in simulated wounds, the growth of these bacteria over time and the ability of the device to provide longitudinal monitoring of bacterial growth in wounds. The device can offer critical information on the biodistribution of bacteria on the surface of the wound that may be useful for directing bacterial sampling and tissue biopsies. Observe, in d) and f), the intense green fluorescence signal of the endogenous collagen at the edge of the pork sample.

This example demonstrates the use of the device for real-time detection of biological changes in connective tissue and bacterial growth based only on autofluorescence, which suggests a practical ability of the device to provide longitudinal monitoring of bacterial growth in wounds.

Reference is now made to Figure 6, which shows examples of the device used for the detection of autofluorescence of connective tissues (e.g., collagen, elastin) and bacteria on the muscular surface of a sample of pork, a) sample that the white light image of the pork used for the tests shows no obvious signs of microbial bacterial contamination or deterioration. However, as seen in b), images of the same area with the device under blue / violet light excitation revealed a bright red fluorescent area of the muscle indicating the potential for bacterial contamination compared to the adjacent side of the muscle. At the edge of the skin an autofluorescence of extremely bright green collagen can also be observed. In c), the device was used to surgically interrogate suspicious red fluorescence to provide a directed biopsy for subsequent pathology or bacteriology. Also note the ability of the device to detect fluorescence contamination (arrow) of the surgical instrument (e.g., forceps) during surgery. In d), the device was used to direct the fluorescence spectroscopy collection using a fiber optic probe from an area suspected of being infected by bacteria (the box shows the device being used to direct the spectroscopy probe into the same area of the red fluorescent muscle in b, c). e) shows an example of the device used to detect contamination by several thin films of bacteria on the surface of the extruded polystyrene container in which the meat sample was kept. The autofluorescence of the bacteria appears as green and red fluorescence streaks under violet / blue excitation light of the various bacterial species previously applied to the meat. Therefore, the device is capable of detecting bacteria on non-biological surfaces where they are hidden under the observation of conventional white light (as in a).

In addition to the detection of bacteria in wounds and on the surface of the skin, the device was also able to identify suspicious areas of muscle tissue, which can then be interrogated by surgery or directed biopsy for pathological verification, or by other optical means such as fluorescence spectroscopy using a fiber optic probe. In addition, contamination by several bacteria was detected on the surface of the extruded polystyrene container in which the meat sample was maintained. The autofluorescence of the bacteria appears as green and red fluorescence streaks under violet / blue excitation light of the various bacterial species previously applied to the meat.

To determine the autofluorescence characteristics of bacteria growing in culture and in simulated skin wounds, hyperspectral / multispectral fluorescence images were used to quantitatively measure the fluorescence intensity spectra of the bacteria under blue / violet light excitation. Reference is now made to Figure 7. In Figure 7, the device was used to detect the fluorescence of bacteria that grow on agar plates and on the surface of a simulated wound in pork, as discussed above for Figures 4 and 5. Bacterial autofluorescence was detected in the green and red wavelength intervals using the device in the culture (a) and in meat samples (d). Hyperspectral / multispectral images were used to obtain images of the bacteria (E. Coli) in the culture (b) and to measure the quantitative spectra of fluorescence intensity of the bacteria (red line: porphyrins, green, cytoplasm, blue, background agar) (c). The red arrow shows the 635 nm peak of porphyrin fluorescence detected in bacteria. The hyperspectral / multispectral image also confirmed the strong green fluorescence (*, right square in d) of P. aeuginosa (with low porphyrin fluorescence, yellow line in f) compared to E. coli (left square in d) where a significant porphyrin red fluorescence was detected e) and g) show the color-encoded hyperspectral / multispectral images corresponding to P. aeruginosa and E. coli, respectively, of the meat surface after 2 days of growth (incubated at 37 ° C); and f) and h) show the corresponding color-coded fluorescence spectroscopy. In i), excitation-emission matrices (EEM) were also measured for the various bacterial species in solution, demonstrating the ability to select the optimum bandwidths of excitation and emission wavelengths for use with optical filters in The imaging device. The EEM for E. coli shows a strong green fluorescence and an important red fluorescence of the endogenous bacterial porphyrins (arrow).

This example shows that the bacteria emit green and red autofluorescence, with some species (eg, pseudomonas aeruginosa) that produce more of the former. Escherichia coli produced significant red autofluorescence from endogenous porphyrins. Such intrinsic spectral differences between bacterial species are significant because they can provide a means to differentiate between different bacterial species using autofluorescence alone. Excitation-emission matrices (EEM) were also measured for each of the bacterial species used in these pilot studies, which confirmed that under excitation with blue / violet light, all species produced a significant green and / or red fluorescence, this Last produced by porphyrins. The spectral information derived from excitation-emission matrices can help optimize the selection of excitation and emission wavelength bandwidths for use with optical filters in the imaging device to allow differentiation of ex -bacterial species ex live and in vivo. In this way, the device can be used to detect subtle changes in the presence and quantity of endogenous connective tissues (e.g., collagens and elastins), as well as bacteria and / or other microorganisms, such as yeasts, fungi and mold in the wounds and surrounding normal tissues, based on unique autofluorescence signs of these biological components.

In addition to prodrugs that improve fluorescence, advances in medicine have allowed the widespread use of fluorescent biomarkers to diagnose diseases at the molecular level. Accurate measurement of the fluorescent biomarker signal in biological tissues can be a critical parameter for obtaining biomolecular information on disease progression and response to treatment, but historically it has posed a significant challenge. To date, this type of advanced molecular imaging has not been reported for wound care. With the use of the device described here, it may be possible to obtain images and monitor said biomarkers for diagnostic purposes.

Images of wound models using exogenous contrast agents

When used to evaluate wounds, tissue autofluorescence imaging can detect relative changes in the remodeling of connective tissue during wound healing, as well as the early presence of bacteria that contaminate, colonize and / or infect wounds (including, among others , bacteria-induced production of exudate and inflammation of the wound). When most wounds are illuminated with violet / blue light, endogenous tissues in the connective tissue matrix (e.g., collagen and elastin) emit a characteristic strong green fluorescent signal, while endogenous bacteria emit a signal of Unique red fluorescence due to the production of endogenous porphyrins. These bacteria include, but are not limited to, common species that are normally found at wound sites (e.g., staphylococcus, streptococcus, E. coli and pseudomonas species ). By using autofluorescence, critical wound information is obtained in real time to provide a means of early detection of key biological determinants of the health status of the wound, which can help stratify patients for optimized care and treatment. of the wound.

The prodrug aminolevulinic acid (ALA) induces the formation of porphyrin in almost all living cells. Many species of bacteria exposed to ALA can induce protoporphyrin IX (PplX) fluorescence [Dietel et al., (2007). Journal of Photochemistry and Photobiology B: Biology. 86: 77-86]. Use ^The ultra low to induce the formation of PpIX in bacteria doses investigated and, therefore, increase the emission of red fluorescence, to increase the contrast of fluorescence from red to green bacteria with imaging device. The device was used to obtain images of live bacterial cultures (staphylococcus aureus, grown on agar plates for 24 h before imaging) using blue / violet excitation light, as seen in Figure 8, which demonstrates the device which is being used in a bacteriology / culture laboratory.

In a), the device was used to obtain images of live bacterial cultures (staphylococcus aureus, grown on agar plates for 24 h before imaging) under white light (circles). In b), the violet / blue excitation light reveals red bacterial autofluorescence, which can be distinguished from the weak green background autofluorescence of the agar growth medium. In c), to increase the red to green fluorescence contrast of staphylococcus aureus against the background agar, an ultra-low dose (~ 20 pg / ml) of the aminolevulinic acid photosensitizer (ALA, in phosphate buffered saline) is usually used in photodynamic therapy (PDT), some of the colonies on the agar plate (identified as "ALA" in the circles) were topically added, while the rest of the agar plate was negative for ALA. After 30 minutes of incubation at 37 ° C, the device was again used to obtain images of the agar plate under excitation with blue / violet light, thus revealing a significant increase in the red fluorescence (of protoporphyrin IX PplX induced by ALA,) of staphylococcus aureus bacteria , compared to colonies (square) that did not receive ALA. Comparison of b) with c) shows that the addition of ALA may be beneficial for the increase in bacterial fluorescence. d) shows the RBG image of c) with the green fluorescence of the agar plate removed, which reveals the increase in red bacterial fluorescence in the colonies of s. aureus treated with ALA. This demonstrates the ability of the device to exploit the use of contrast agent strategies to increase the signal in the background for the sensitive detection of bacteria, for example, in wounds. The time required for ALA to increase PpIX fluorescence to detectable levels was 30 minutes, suggesting that this technical approach may also be clinically practical. Moreover, this also demonstrates that the device can be used to obtain photosensitizing fluorescence images (e.g., PpIX) in bacteria, which grow in culture or in wounds of patients for subsequent treatment with PDT.

After 30 minutes of incubation of staphyloccous aureus ~ 20 pg / ml of ALA at 37 ° C, a significant increase in the red fluorescence of the bacteria was detected, compared to those (square) colonies that did not receive ALA. This demonstrates the ability of the device to exploit the use of contrast agent strategies to increase the signal in the background for the sensitive detection of bacteria, for example, in wounds. The time required for ALA to increase the PpIX fluorescence of bacteria in culture to significant levels was approximately 0.5 h, suggesting that this technical approach may also be clinically practical. Tests on simulated meat samples contaminated with bacteria revealed similar results to those obtained from bacterial cultures. Topical application of 0.2 pg / ml of ALA by spraying on wounds in pigskin produced a dramatic increase in the contrast of bacterial porphyrin red fluorescence approximately 2 h after administration of ALA. This can allow the detection of bacterial contamination with fluorescence images at the wound sites and any other part of the skin surface, which was previously hidden under white light images, as demonstrated with reference to Figures 9 and 10.

Figure 9 shows examples of use of the device for the detection of autofluorescence of connective tissues and varies the bacterial species on the skin surface of a sample of pork. To determine if the intensity of the bacterial fluorescence can be increased to obtain images with the device, the non-toxic aminolevulinic acid (ALA) prodrug (~ 0.2 mg / ml PBS) was applied topically to the skin surface by spraying using an atomizing bottle The meat sample was then placed in a light-tight incubator at 37 ° C for approximately 3-4 h until white light and fluorescence imaging was performed using the imaging device.

Referring to Figure 9, a) shows white light images of the pork used for the test. In b), several bacterial species were applied to small skin incisions [(1) streptococcus pyogenes, 2) serratia marcescens, 3) staphylococcus aureus, 4) staphylococcus epidermidis, 5) escherichia coli, and 6) pseudomonas aeruginosa)] . Under violet / blue excitation light, the device shows bacterial autofluorescence (green and red fluorescence at the wound sites). The presence of endogenous porphyrin red fluorescence can also be observed in other areas of the skin surface (red arrow). The fluorescence of the bright collagen can also be observed at the edge of the sample (blue arrow). Bacteria on the surface of the extruded polystyrene container containing the meat sample are also detected by autofluorescence with the device, but are hidden under white light (left panel). This indicates that the device can be used to detect and form images of the presence of bacteria or microbes and other pathogens on a variety of surfaces, materials, instruments (e.g., surgical instruments) in hospitals, chronic care centers, asylums for the elderly and other healthcare settings where pollution can be the main source of infection. The device can be used together with the detection, identification and conventional enumeration of indicator organisms and pathogen strategies.

In c), the non-toxic aminolevulinic acid (ALA) prodrug (0.2 mg / ml) was applied topically to the surface of the skin to determine if bacterial fluorescence can be improved. The result, approximately 1 h after administration of ALA, was a dramatic increase in the fluorescence of bacterial porphyrin (bright red fluorescence) both in skin tissue and at wound sites, as well as in the surface of the Extruded polystyrene container in which the meat sample (arrows) was kept. This illustrates the possibilities for biopsy orientation by fluorescence imaging guide, and the use of the device for the detection and subsequent treatment of infected areas using PDT, for example.

Figure 10 shows examples of the use of the device for the detection of bacterial infection with fluorescence contrast in a sample of pork, a) shows the white light image of pork. Several bacterial species were applied to small incisions made in the skin (arrow). In b), the non-toxic aminolevulinic acid (ALA) prodrug (0.2 pg / ml) was applied topically by spraying using a common spray bottle and the imaging device was used to obtain images of the red fluorescence protoporphyrin IX (PpIX) induced by resulting ALA. Images of the skin surface (approximately 2 h after administration of ALA) with violet / blue light (405 nm) resulted in a dramatic increase in the contrast of red fluorescence of bacterial porphyrin indicating the presence detection of bacterial contamination with fluorescence images in the incisions of the simulated surgical wound (arrows) and in other parts of the skin surface, which were previously hidden under light images white (circle in a and b). Note that some areas of the skin surface that were not exposed to oxygen because the sample was placed "under the skin" in the container does not emit bright red fluorescence, possibly due to suspected oxygen dependence for bacterial production of PpIX. Some bacteria produce bright green autofluorescence signals that are also detected by the device. In c), in another sample of pork, bacteria hidden under white light (circle) images are easily detected using autofluorescence images only (box). However, as shown in d), topical application of low dose ALA caused a dramatic increase in bacterial fluorescence after 2 h, demonstrating the utility of exogenous prodrugs as fluorescence image contrast enhancing agents for Better detection of bacterial contamination. Note the bright green autofluorescence of endogenous collagen and elastins in the connective tissues in the sample. In e) and f), ALA-induced fluorescence allowed the detection of hidden bacteria on the surface of the skin (circles), which offers the possibility of directing an image-guided biopsy and the use of the device for detection and treatment posterior areas infected by PDT, for example.

The device can also be used together with exogenous pro-drug agents, which include, among others, ALA that is approved by the FDA for clinical therapeutic indications, to increase the endogenous production of porphyrins in bacteria / microorganisms and thus increase signal intensities. of unique "porphyrin" fluorescence emanating from these bacteria to improve the detection sensitivity and specificity of the device. Therefore, the device can be used to conveniently visualize photosensitizer-induced fluorescence (e.g., PpIX) in bacteria, which grow in culture or in patient wounds for subsequent sampling or image-guided guided biopsy, or treatment with photodynamic therapy (PDT) [Jori et al. Lasers Surg Med. June 2006; 38 (5): 468-81; Dougherty et al. (1998) J. Natl. Inst. Cancer 90, 889-905; Carruth (1998) Int. J. Clin. Pract. 52, 39-42; Bissonnette et al. (1997) Dermatol. Clin 15, 507-519]. PDT can provide a complement to current antibiotic treatment or an alternative in which antibiotics no longer work (e.g., drug-resistant strains). The available evidence suggests that strains resistant to multiple antibiotics are as easily destroyed by PDT as naive strains, and that bacteria may not easily develop resistance to PDT. This can be vital for the treatment of wounds in patients undergoing cancer therapy, HIV patients demonstrating resistance to antibiotics and the elderly with persistent oral infections [Hamblin et al. (2004) Photochem Photobiol Sci. 3: 436-50].

The device can be used to detect bacteria or microorganisms in the wound and surrounding normal tissues using low-light blue / violet excitation light / illumination, but it can also be used immediately afterwards to destroy them, for example, using PDT or other therapies . Through the use of high-power red light / excitation light, endogenous porphyrins in bacteria or microorganisms can be destroyed at the site of injury by PDT. Therefore, this device may have the ability to serve as an all-in-one non-invasive or non-contact "search and treat" instrument for clinical wound care. In addition, once bacteria or microorganisms are detected, the device can be used to treat and / or disinfect the wound site with PDT, and then an image of the site can be re-taken to determine the effectiveness of PDT treatment. In some embodiments, the device can only be used for detection / diagnosis purposes and cannot perform any therapeutic treatment by itself. The device can be used continuously until the entire wound and surrounding normal tissue have been disinfected, and the wound can be subsequently monitored longitudinally as part of the conventional clinical follow-up. The fluorescence images of the device can be used to determine the biodistribution of the photosensitizer or PDT photoproducts [Gudgin et al. (1995) J. Photochem. Fotobiol B: Biol. 29, 91-93; Konig et al. (1993) J. Photochem. Fotobiol B: Biol. 18, 287-290], since most of these are intrinsically fluorescent, and therefore the device can serve as a means to direct the PDT treatment light. Therefore, the device can guide, by imaging, the integrity of the PDT treatment. Similarly, the device can be used to guide other therapies.

Since it is known that some photosensitizers are photobleached [Jongen et al. (1997) Fis. Med. Biol. 42, 1701 1716; Georgakoudi et al. (1997) Photochem. Photobiol 65, 135-144; Rhodes et al. (1997) J. Investig. Dermatol 108, 87-91; Grossweiner (1986) Lasers Surg. Med. 6, 462-466; Robinson et al. (1998) Photochem. Photobiol 67. 140 149; Rotomskis et al. (1996) J. Photochem. Photobiol B: Biol. 33, 61-67], the fluorescence imaging capability of the device can be used to determine the degree or rate of photobleaching of the photosensitizer. This information may be useful for optimizing PDT dosimetry [Grossweiner (1997) J. Photochem. Photobiol B: Biol. 38, 258-268] in order to ensure adequate treatment of the disease, while minimizing damage to surrounding normal tissues. The device, with excitation light sources that can be selected for specific wavelengths and excitation intensities, in one embodiment, can also be used to supply the light for PDT combined with any commercially available and / or experimental PDT photosensitizer. Therefore, it may be useful in the clinical indications of existing PDT (e.g., for the surface of the skin or hollow organs) and / or in the field of commercial / academic research and the development of future photosensitive agents of PDT, both preclinically and clinically.

Figure 10G shows an example of the use of the device to monitor the response of bacteria to photodynamic therapy (PDT). Porcine tissues were prepared ex vivo in Petri dishes and contaminated with bioluminescent Staphylococcus aureus (BL) 24 h before BL and fluorescence image of samples using the device Bioluminescent and corresponding fluorescence images were made in a, d) uncontaminated muscle tissues, and b, e) contaminated with SA before and after PDT. Note that Staphylococcus aureus produced a fluorescent red color (white arrow on e). PDT was performed on the sample of meat contaminated with bacteria (marked with a yellow circle) by incubating the sample with a common photosensitizer called methylene blue (MB) for approximately 30 minutes, followed by the removal of excess MB (and rinse with PBS) and subsequent exposure to a light source of approximately 670 nm (here an LED matrix) for about 10 minutes at ~ 10 J / cm2 to cause photodynamic treatment. Comparison of BL intensity scales in b) and c) shows a marked decrease in BL intensity in the sample of meat treated after PDT (e.g., PDT has destroyed a measurable proportion of bioluminescent bacteria , thus decreasing the intensity of the BL signal) and the fluorescence characteristics (e.g., intensity and biodistribution) of the Staphylococcus aureus bacteria (red color) can be seen using the manual imaging device after the PDT. Note that the intense green fluorescence in the meat sample (pink arrow in e) was caused by an involuntary cross contamination of the meat sample by Pseudomonas aeruginosa no BL during the experiment (confirmed by bacteriology), and the device detected this . These data suggest the use of the device to monitor the use of PDT for the treatment of bacterial contamination in biological (and non-biological) samples. (405 nm excitation; 490-550 nm and> 600 nm emission).

This device can be used as an imaging device and / or supervision in clinical microbiology laboratories. For example, the device can be used to obtain quantitative images of bacterial colonies and quantify colony growth in common microbiology assays. Fluorescence images of bacterial colonies can be used to determine growth kinetics.

Obtaining images of blood in wounds

Angiogenesis, the growth of new blood vessels, is an important natural process required to heal wounds and to restore blood flow to tissues after an injury or trauma. Angiogenesis therapies, which are designed to "activate" new hair growth, are revolutionizing medicine by providing a unified approach to the treatment of paralyzing and life-threatening conditions. Angiogenesis is a physiological process required for wound healing. Immediately after the injury, angiogenesis is initiated by multiple molecular signals, which include hemostatic factors, inflammation, cytokine growth factors and interactions between the cell and the matrix. The new capillaries proliferate through a cascade of biological events to form granulation tissue in the wound bed. This process can be maintained until the terminal stages of healing, when angiogenesis is stopped by decreased levels of growth factors, inflammation resolution, stabilized tissue matrix and endogenous angiogenesis inhibitors. Defects in the angiogenesis pathway alter granulation and delay healing, and these are evident in chronic wounds [Tonnesen et al. (2000) J Investig Dermatol Symp Proc. 5 (1): 40-6]. By illuminating the tissue surface with selected narrow wavelength bands (e.g., blue, green and red components) of light or by detecting the reflectance of white light in several narrow bandwidths of the visible spectrum (e.g. ., selected wavelengths of maximum absorption of the white light blood absorption spectrum), the device can also be used to obtain images of the presence of blood and microvascular networks within and around the wound, including the surrounding normal tissue, which also reveals areas of erythema and inflammation.

Reference is now made to Figure 11. The device can use individual optical filters (e.g., 405 nm, 546 nm, 600 nm, / - 25 nm each) to demonstrate the possibility of obtaining blood and microvasculature images in wounds White light images of a wound can be collected with the device and then with the device, equipped with a triple band pass filter (e.g., 405 nm, 546 nm, 600 nm, / - 25 nm each ), placed in front of the imaging detector, can visualize the narrow bandwidths separated from the reflected light components of blue (A), green (V) and red (R) of the wound. These wavelength bands can be selected based on the maximum absorption wavelengths of the blood, which contain oxygenated and deoxygenated hemoglobin, in the visible light wavelength range. The resulting images can produce the relative absorption, and therefore the reflectance, of the light visible by the blood in the field of vision. The resulting "blood absorption" image produces a high contrast image of the presence of blood and / or microvascular networks in the wound and in the surrounding normal tissues. The physician may select the appropriate set of optical filters for use with the device to obtain images of the blood and / or the microvascular distribution in the wound and combine this information with one or both autofluorescence images and images with exogenous contrast agents. This may provide a global set of information about the wound and surrounding normal tissues at the morphological, topographic, anatomical, physiological, biological and molecular levels, which may not currently be possible within the conventional practice of wound care.

Figure 11 shows examples of the device used to obtain images of blood and microvasculature in wounds. The device was used to obtain images of a piece of filter paper stained with blood (a) and the ear of a mouse during surgery (b). White light images of each specimen were collected using the imaging device, in non-fluorescent mode, and then equipped with a triple band pass filter placed in front of the imaging detector (405 nm, 546 nm, 600 nm, / -25 nm each) to obtain images of the narrow bandwidths separated from the components of reflected light blue (A), green (V) and red (R) of the specimens. These wavelength bands were selected based on the maximum blood absorption wavelengths in the wavelength range of visible light (box in a) shows the spectral absorption profile of oxygenated and deoxygenated hemoglobin in the blood. This shows that by using a simple multiband transmission filter, it is possible to combine the three images A, V, R into a single "white light equivalent" image that measures the relative absorption of light by blood in the field of vision. The resulting "blood absorption" image produces a high contrast image of the presence of blood containing oxygenated and deoxygenated hemoglobin. The device can be used with narrower bandwidth filters to obtain images of greater contrast of blood absorption in wounds, for example.

The regulation of angiogenesis over time during wound repair in vivo has been largely unexplored, due to difficulties in observing events in the blood vessels. Although the initial tests of the imaging device were exploratory, simple modification of the existing prototype can allow longitudinal images of dynamic changes in blood supply and microvascular networks to be obtained during the wound healing process in vivo.

Image of the skin and oral cavity

This device may be suitable for obtaining images of the skin, mouth and oral cavity. The device may allow the detection of changes in connective tissue due to minor skin lesions (e.g., cuts, abrasions) and endogenous bacteria that are commonly found in normal skin (e.g., Propionibacterium acnes or P. acnes ).

This device may also be suitable for multispectral imaging and / or monitoring of dental plates, carriers and / or cancers in the oral cavity. The device can be used to detect the presence of plaques, periodontal diseases, carriers and cancers, as well as local oral infections, based on the presence of unique autofluorescence signs in abnormal or neoplastic tissues. The device can use white light, fluorescence, with or without autofluorescence or exogenous fluorescent agents, and reflectance images to provide real-time detection and diagnosis of periodontal diseases, plaques, and carriers and cancers in the oral cavity. The device can record the images for cataloging medical records. Unlike the direct vision approach (i.e., the naked eye) used by an existing product such as the VELscope System, by the Vancouver-based company LED Medical Diagnostics Inc. (LED-MD), the present device can provide images digital and recording of white light, fluorescence and tissue reflectance information.

In dermatology, the device can be used to detect bacteria in normal skin. For example, Figure 12 demonstrates the high resolution autofluorescence image of the normal skin of the faces of patients in whom a clear red fluorescence of the common bacterium Propionibacterium acnes is detected.

Figure 12 shows examples of the use of the device for still images or high-resolution non-invasive video images of the oral cavity and skin surface in patients. As shown in a), the device can be used to obtain images of the mouth and oral cavity. The excitation with blue / violet light excites the autofluorescence of the teeth, which appears as an intense green fluorescence, compared to blood-rich gums. The use of this device can easily detect periodontal disease and caries according to the autofluorescence of the teeth and gum tissues. Red fluorescence at the edge of the lips is detected in Propionibacterium acnes (P. acnes) that is commonly found in the pores of the skin. The red fluorescence is produced by endogenous bacterial porphyrins. Note the detection of P. acnes in individual pores (red arrow) on the lip. Similarly, in b), the red fluorescence of endogenous porphyrins in the normal bacterial fauna of the tongue is easily detected as a bright red fluorescent "blanket" on the surface of the tongue. The device can also be used to detect early cancers in the oral cavity based on differences in optical properties (e.g., absorption, dispersion, autofluorescence) between normal and pre- and neoplastic tissues. The device can be used to "scan" the oral cavity of mucosal cancers, or to determine the effects of antineoplastic therapeutic agents such as PDT or other techniques. The device can also be used to obtain images of the skin surface. In c) -e), the device takes pictures of the skin on the faces of the patients by detecting the autofluorescence produced by the excitation of violet / blue light from the skin surface. The red fluorescence of P. acnes can be easily detected in the regions of the face (e). The device can be used to visualize and / or monitor the potential effects of dermatological interventions (eg, topical creams, drugs and other antibiotics, etc.) on patients' skin. In f) and g), the device was also used to obtain images of minor cuts (arrow, h), scratches and abrasions on the skin of patients, as well as psoriasis on a finger (arrow, i). Under violet / blue light, the device detected tissue autofluorescence of the connective tissue components (e.g., collagen and elastin) of the wound site and surrounding normal skin to produce high-resolution images of subtle skin lesions. P. acnes is the causative agent of acne vulgaris (i.e., pimples) and is a common resident of the pilosebaceous glands of human skin, and is hidden under the visualization of white light. These autofluorescent images were obtained without the need for exogenous agents / drugs and demonstrate the ability of the device to Detect the fluorescence of bacterial porphyrin in the pores of the skin.

Figure 12J shows an example of the use of the imaging device for real-time fluorescence detection of common bacterial flora in the skin. a) The red fluorescence in and around the nose is detected in Propionibacterium acnes (P. acnes) that is commonly found in the pores of the skin. b) The fluorescence image can also be used to detect and monitor more than one bacterial species on the skin at the same time, for example, Propionibacterium acnes appears as a red fluorescent (red arrow) while Pseudomonas Aeruginosa has a bright green color (arrows green). These data suggest the use of the device to distinguish relative concentrations / levels of various bacterial species, determine their biodistributions on the body surface and monitor the response to antibacterial treatments in dermatology and cosmetology applications, c) show an example of a fluorescence image of a culture grown on agar from a smear taken from normal skin on the nose of a healthy volunteer. Bacteriology results showed the presence of Pseudomonas aeruginosa .

Such ability to capture images and document the presence and biodistribution of bacteria on the surface of the skin makes the device potentially useful in the fields of dermatology and cosmetology. For example, the fluorescence image can be performed before, during and after the application of dermatological treatment and / or pharmaceutical / cosmetic formulations (e.g., topical creams, drugs and other antibiotics, skin disinfectants, treatments for acne, etc.) to normal and abnormal skin conditions, which include, among others, scar formation, hyperpigmentation, acne, psoriasis, eczema, skin rashes, etc. Fluorescence / reflectance-guided image tattoo removal (e.g., by surgery or available laser treatments) may also be an option with the device. The device was also used to obtain images of minor cuts, scratches and abrasions on patients' skin and under violet / blue light, tissue autofluorescence of connective tissue components (e.g., collagen and elastin) from the wound site. and the surrounding normal skin helped to detect hidden white light changes in connective tissues during healing of the minor skin wound (as seen in Figure 12 h, i). In addition, the device can also serve as a practical, cost-effective and sensitive image-based tool for the early detection of hidden skin cancers and non-neoplastic (i.e., benign) lesions in a non-invasive manner [Chwirot et al. (1998) Eur J Cancer. 34 (10: 1730-4). The device can be used to provide an image guide for surgical excision of lesions or for PDT. For the latter, fluorescence images can monitor the response of the PDT and determine completion of treatment over time with multiple longitudinal image scans of the affected area.The device can be used in real time to determine the location and biodistribution of the PDT photosensitizer and photobleaching, and this can be mapped into the light image white of the area to be treated for an anatomical comparison Changes in the optical properties between normal and burned diseases or tissues can be detected using the white light and fluorescence imaging capabilities of the device. The device can also be used to visualize, evaluate and monitor the burn healing process longitudinally or determine the response of foot grafts l or temporary skin substitutes in the treatment of burn patients [Bishop (2004) Crit. Care Nurs Clin North Am.

200416 (1): 145-77]. The device can also be used to detect and control radiation-induced late skin damage during the treatment of patients with ionizing radiation [Charles (2007) J Radiol Prot. 27 (3): 253-74].

In addition, the device can be used to obtain images of the mouth and the oral cavity, particularly in the embodiment in which the device is small and compact. Pilot imaging studies showed that the device can detect endogenous bacteria in the oral cavity (e.g., on the surface of the tongue and between the teeth in the gum line), suggesting a use in clinical detection. of caries and periodontal disease [Pretty (2006) J Dent. 34 (10): 727-39]. Additionally, tissue autofluorescence has proven useful in the detection of oral cancers [Kois et al. (2006) Dent Today. 25 (10): 94, 96-7]. The device can be used to detect early cancers in the oral cavity based on differences in optical properties (e.g., absorption, dispersion, autofluorescence) between normal, pre- and neoplastic oral tissues. In addition, the device can be used to "scan" the oral cavity for mucosal cancer and monitor the response to therapy.

In general, the device can be used to visualize and / or monitor targets such as a cutaneous target, an oral target, an otolaryngological target, an ocular target, a genital target, an anal target and any other suitable target in a subject.

Use in malignant wounds

A malignant wound is also known as tumor necrosis, tumor wound, ulcerative neoplastic wound or malignant skin wound. A malignant wound can be an emotional and physical challenge for patients, families and even for the experienced doctor. Tumor and ulcer wounds can be unsightly, smelly and painful. These wounds can be indicators of disease progression and can become infected, which causes a delayed / impaired healing and associated morbidity and, therefore, reduces the quality of life of patients.

Many cancer patients live with the knowledge that their disease is progressive and incurable. For a significant minority of these people, this reality may be present in the form of a skin lesion necrotic, oozing and smelly, which can be a constant physical reminder of disease progression (Mortimer PS. in: Doyle et al. Editors. Oxford Textbook of Palliative Medicine (2nd ed.). Oxford: Oxford University Press, 1998, 617-27; Englund F. RCN Contact 1993; Winter: 2-3). These lesions are commonly known as "tumor wounds," and the term "tumor" refers to a malignant process of ulcerative and proliferative growth (Grocott P. J Wound Care 1995; 4 (5): 240-2). Lesions that have a predominantly proliferative growth pattern can become a nodular "fungus" or "cauliflower" lesion, while an ulcer lesion will produce a crater-like wound (Grocott P. J Wound Care 1999 , 8 (5): 232-4; Collier M. Nurs Times 1997; 93 (44): Suppl. 1-4). Such lesions may also present with a mixed appearance of areas in proliferation and ulceration (Young T. Community Nurse 1997; 3 (9): 41-4).

A malignant wound can develop in one of the following ways:

• As a result of a primary skin tumor such as squamous cell carcinoma or melanoma. • Through direct invasion of the skin structures by an underlying tumor, for example, breast cancer or hematologic malignancies such as cutaneous T-lymphocyte lymphoma (fungoid mycosis).

• Of the metastatic spread of a distant tumor. Metastasis can occur along the tissue, capillary or lymphatic planes.

Malignant wounds are often difficult to treat due to their location, smell, excessive exudates and the propensity to bleed. Each malignant wound may be unique in its appearance and presentation symptoms. Common symptoms associated with malignant wounds include bad smell, excessive exudates, infection, bleeding, maceration and excoriation of perilesional skin, pruritus, pain, poor aesthetics and aesthetic effects of dressings. Currently, the focus of attention is mainly holistic and palliative, with the aim of controlling the symptoms at the site of the wound and reducing the impact of the wound on the patient's daily life, mainly by identifying bacterial / microbial infections and supervision of signs of healing. Unless the pathology is controlled, these wounds are not expected to heal.

The described device may be useful for performing a clinical evaluation of such wounds (eg, physical and biological examination). The device can provide: a means of complete wound assessment based on images at baseline and at regular intervals throughout the treatment (i.e. longitudinal supervision), wound assessment including location, wound size, color, type and amount of any discharge or drainage, series of white light images (e.g., for color changes) and fluorescence (e.g., for structural, cellular, biological and molecular tissue changes) of chronic malignant wounds, and It can provide an evaluation of any signs and symptoms of infection in real time, which would affect the planning and effectiveness of the treatment. The device can be integrated into current clinical practice for the evaluation and care of such malignant wounds. Image of exogenous fluorescence contrast agents

The development of highly efficient analytical methods capable of exploring biological systems at the system level is an important task that is required to meet the requirements of the emerging field of systems biology. Optical molecular imaging is a very powerful tool for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vivo. There have been several recent advances in optical molecular images, such as the development of molecular probes that make the images brighter, more stable and more biologically informative (e.g., FPs and semiconductor nanocrystals, also referred to as quantum dots) , the development of image approaches that provide greater resolution and greater penetration into tissues, and applications to measure biological events from the molecule to the organism level. These advances can also be applied to the diagnosis of diseases (eg, wound care) and pharmaceutical systematic detection. However, current fluorescence imaging devices are large, complicated and involve expensive optical components and very sensitive camera detectors, which makes these systems extremely expensive. The device developed here offers an alternative to these cost limitation systems for preclinical or research studies, as well as a possible clinical translation of such methods.

Reference is now made to Figure 13. The device was also used to obtain images of the animal for general observation under fluorescence to determine the degree of fluorescence of the BPD photosensitizer on the entire surface of the skin. Figure 13 demonstrates the utility of the device for real-time imaging and sensitive detection of exogenous fluorescence contrast agents in vivo (e.g., quantum dots, QDots). In a), the device was used to obtain images of exogenous fluorescence contrast agents in a sacrificed rat that carried metastatic human breast tumor cells in the bone in the hind leg. The rat was previously injected with a fluorescence photosensitizer called benzo-porphyrin derivative (BPD) for an unrelated photodynamic therapy experiment. Two separate fluorescent semiconductor nanoparticle solutions (here, QDots) were administered to the rat, each emitting fluorescence in solutions at 540 (+/- 15) nm and 600 (+/- 15) nm by subcutaneous injection in the hind leg. left. The injections were approximately 1 cm apart. The device was then used to obtain images of the entire rat body using a blue / violet excitation light. The evaluated skin appeared in red, and this was probably due to the combination of the fluorescence of the benzo-porphyrin-derived photosensitizer (BPD) administered to the rat before the experiment, which was for subsequent PDT, as well as contamination of dust and food from the cage in which the rat was housed.

With reference still to Figure 13, in b) the fluorescence of the green and red QDots (inset) was easily detected under the skin at the injection site, with the red QDots emitting the brightest signal, due to the higher Red light penetration into the tissue. c) shows an enlarged image of the rear leg shown in b). The device was able to detect multiple fluorescence contrast agents simultaneously together with the autofluorescence of the background tissue with sufficient signal to noise (green and red arrows) to allow its use in preclinical and clinically expected fluorescence imaging of fluorescence contrast agents molecularly directed multiplexed in vivo. Note that the green fluorescence is weaker than the red one because both the violet / blue excitation light and the subsequent green QDot fluorescence are preferably absorbed by the blood and the red QDot fluorescent light has a greater depth of penetration through the tissue . In d), the device was also used to obtain images of the animal for general observation under fluorescence to determine the degree of fluorescence of the BPD photosensitizer on the entire surface of the skin. The device may also be useful for guiding intravenous injections using needles when detecting superficial blood vessels under the skin. Therefore, the device can be used to detect fluorescent tumors, such as those that are transfected with fluorescent proteins and cultured subcutaneously in an xenograft or orthotopic model. Thus, the device can be used to visualize multiple wound healing and / or infectious biomarkers using multiplexed exogenous fluorescent molecular targeting agents (e.g., for in situ imaging based bacteriology ).

To improve the use of fluorescence contrast agents in preclinical research and, finally, for the clinical translation of optical molecular imaging technologies, it is desirable to be able to differentiate and identify several fluorescent agents relatively quickly. In e) and f), the device was also used as a means to identify relatively quickly what fluorescence contrast agents were in the syringes before injection, which was not possible under conventional white light, demonstrating the utility of the device as cost-effective fluorescence imaging guided technology to provide useful information quickly during fluorescent imaging guided and / or PDT procedures, in which fluorescent compounds are commonly used, possibly even in emerging wound care techniques.

Fluorescence guided image surgery

An emerging area is the use of fluorescence imaging for systematic diagnostic detection and image-guided surgery. Overcoming the limitations of conventional white light surgery, fluorescence imaging can be used to aid in the surgical resection of tumors in vivo based on fluorescence (e.g., autofluorescence or fluorescence of exogenous directed / non-directed contrast agents ), as well as to verify the integrity of the tumor removal (e.g., clear margins). Fluorescence-guided image surgery has shown improvements in survival, preclinical and clinically [Bogaards et al. (2004) Lasers Surg Med. 35: 181-90]. For example, during exploratory surgery in a rat, the device can provide conventional white light images of the surgical field.

Reference is now made to Figure 14. Several tests were performed to demonstrate the usefulness of the device for fluorescent imaging guided surgery in small animals. Exploratory surgery was performed on a female rat euthanized using the imaging device. Figure 14 shows examples of the use of the device for fluorescence-guided surgery using imaging agents. During the exploratory surgery, the device provided conventional white light images of the surgical field, here, the abdomen of a female rat (a). The surgeon used the device's display screen to guide the procedure, easily and quickly switching between white light and fluorescence mode. In b), using violet / blue excitation light, the device provided an added contrast between different types of tissues, which was not possible during imaging with white light. For example, connective tissues appeared in the bright green fluorescent (green arrow), while the skin surface (with the red BPD fluorescent photosensitizer) appeared red (red arrow), and the QDots previously injected into the hind leg They appeared in a bright red (blue arrow). The fluorescence image was used to detect contamination of surgical instruments and equipment (e.g., gauze, tapes, blankets, etc.) during the surgical procedure. In c), the device also proved useful in detecting a dirty / contaminated surgical gauze during the procedure. Compared to the conventional white light under which all the gauze seemed clean, the gauze used to clean the skin and the surgical field during surgery seemed red fluorescent (left) compared to a clean gauze (right).

The device was also used for real-time detection of exogenous fluorescent contrast agents (e.g., for tracking of labeled cells and the destination in in vivo experiments , for tissue engineering studies in regenerative medicine, etc.) in An animal model. For this, during surgery, the device was used in fluorescence mode to visualize the presence of red fluorescent QDots injected into the heart muscle and in the lungs of the rat (d). Under the violet / blue excitation light, red QDots can be easily detected in the heart (e) and in the lungs (f), which appear dark due to the high concentration of blood in these organs, demonstrating the usefulness of the device for guiding and targeting biopsies or microsurgical procedures, especially those aimed at detecting and removing cancers (e.g., using autofluorescence or improving fluorescence contrast) Observe the bright red autofluorescence detected by the device from digested food material in the colon. In g), the device demonstrated its utility in obtaining images of fluorescent tumor ghosts commonly used in the investigation of images of small animals. Solid spherical tumor ghosts doped with fluorescent dye were prepared in different sizes and placed in the surgical field to demonstrate the ability of the device to provide fast "high contrast" fluorescence images in small animal cancer models.

These results show that the device can be useful for detecting sub-mm lesions with fluorescence guidance, which may be useful for conducting biopsies or microsurgical procedures, especially those aimed at the detection and removal of cancers (e.g., using autofluorescence or fluorescence contrast enhancement). The device may also be useful in obtaining images of fluorescent tumor ghosts commonly used in the investigation of images of small animals.

Figure 15 shows examples of the device being used for video recording of the rat-guided high resolution fluorescence imaging surgery in Figure 9. The device may be able to provide still digital images and films taken with light Conventional white (LB) (a) and fluorescence (FL) (b), which can be easily changed. In this case, the device was used to capture digital films of a surgical procedure in a rat using white light and fluorescence images. The surgeon used the digital display screen of the device to guide the entire surgical procedure using fluorescence in which white light did not provide adequate information. In c) -e), for example, under excitation with blue / violet light, the fluorescence image gave the surgeon a significant image contrast between different types of tissues. Blood vessels can be seen clearly under fluorescence and connective tissues can be distinguished from the gastrointestinal tract. The digested food material can also be distinguished. The device can provide a real-time imaging solution for an image-guided surgical intervention or a biopsy that allows the surgeon to make critical judgments during the procedure. Digital image capture and / or surgery film can allow retrospective analysis of the procedure for patient health records and future training in medical staff skills. The device can also record audio during the surgical procedure, allowing you to collect a complete record of each procedure. The usefulness of the device was also demonstrated as a very useful tool for minimally invasive microsurgery guided by images in animals, and potentially in human procedures.

Figure 16 shows examples of the device used for guided surgical resections with tissue autofluorescence imaging in a mouse heart attack model (a). During the exploratory surgery, the device provided conventional white light (LB) images of the open surgical field, in this case, the abdomen of the mouse (b). The surgeon used the device's display screen to guide the procedure, easily and quickly switching between white light and fluorescence mode. Using violet / blue excitation light, the device provided a high contrast between different types of tissues, which was not possible during imaging with white light (c). For example, several internal organs were visualized using high resolution autofluorescence images. In d), images of the intact animal can be obtained with fluorescence before and during surgery (e).

Figure 17 shows examples of the device used for non-invasive real-time image-guided autofluorescence surgery of a mouse brain. During the exploratory surgery, the device provided conventional white light (LB) images of the open surgical field (a); In this case, you can see the skull of the mouse. The surgeon used the device's display screen to guide the surgical procedure, easily and quickly switching between LB mode and fluorescence (FL). b) shows the view of the surgical field (in this case, with the skull intact) provided by the imaging device under tissue autofluorescence. Note that the surgical area is dark, mainly due to the absorption of violet / blue excitation light and the resulting autofluorescence caused by blood. The snout and eyes have a bright red fluorescent color compared to the bright green fluorescence of the fur. c) shows the surgical field with the skull base removed under LB, while d) shows the autofluorescence image of the surface of the brain using the blue / violet excitation light imaging device. Injection of an exogenous contrast agent (in this case, red fluorescent quantum dots) directly into the right hemisphere of the brain produces a bright red fluorescence (arrows) (e). This demonstrates the usefulness of the device to obtain images of the fluorescence contrast agents, specifically for guided high resolution fluorescence imaging surgery.

Use in Clinical Care

Although the current practice of wound treatment aims to reduce the morbidity and mortality of wounds in patients, a limitation is the availability of resources for medical care. The potential of incorporating telemedicine technology into wound care needs is currently being explored. Wound care is a representation of the care of chronic and debilitating conditions that require specialized long-term care. The greatest effect of improving living conditions and advances in care Worldwide doctors have led people to live longer. Therefore, the percentage of elderly people in the world and people with chronic medical conditions that require medical attention is increasing. With the escalation in the costs of medical care and the industry's drive towards outpatient care, this is part of the crisis of medical care that demands immediate attention.

The present device can provide biologically relevant information about wounds and can exploit the emerging telemedicine infrastructure (e.g., E-health) to provide a solution for mobile wound care technology and can have a great impact on treatment. of the medical care of the wound. Wound care represents a large percentage of home visits by nurses and health workers. Despite best practices, some wounds do not heal as expected and require the services of a clinical specialist. The device described in this case may allow access to specialized clinical resources to help treat wounds from the comfort of the patient's home or chronic care center, which reduces travel time for clients, increases availability for patients. specialists in clinical wounds and can reduce costs for the health system.

Different uses of the imaging device for wound evaluation, supervision and care management have been discussed. The device can be used to detect and monitor changes in connective tissues (e.g., collagen, elastin) and blood / vascular supply during the wound healing process, monitor tissue necrosis and exudate in fluorescence-based wounds, detect and diagnose wound infections, even potentially indicating "clinically significant" critical categories of the presence of bacteria or microorganisms (eg, to detect contamination, colonization, critical colonization and infection) on the surface and in the depth of the wounds [Kingsley, Ostomy Wound Manage. July 2003; 49 (7A Suppl.): 1-7], provide topographic information of the wound and identify the margins of the wound and surrounding normal tissues. Fluorescence and tissue reflectance image data can be "mapped" on the white light images of the wound, allowing visualization in the wound and surrounding normal tissues of the essential biochemical and photobiological information of the wound (p e.g. fluorescence), which has not been possible to date. Real-time wound images can be made over time to monitor changes in wound healing and, potentially, to monitor the effectiveness of treatments by providing useful information about the underlying biological changes that are occurring at tissue / cell level (eg, matrix remodeling, inflammation), infection and necrosis). This can provide quantitative and objective information about the wound for the detection, diagnosis and monitoring of treatment in patients. In particular, the device can be used to monitor and / or track the effectiveness of the therapy at the biological level (e.g., at the bacterial level), which can provide more information than just monitoring the macroscopic / morphological aspect using white light.

The device can provide non-invasive real-time guided image biopsy guidance, clinical procedure guidance, tissue characterization and can allow image-guided treatment using conventional and emerging modalities (e.g., ^p D ^t ). In addition, the use of the imaging device can be used to correlate the biological and molecular critical information of the wound obtained by fluorescence (eg, autofluorescence of endogenous tissue and / or administration of biomarker-directed fluorescence contrast agents). exogenous molecules) with current and emerging clinical evaluation and treatment guidelines for wound care, such as the NERDS and STONES guidelines proposed by Sibbald et al. (Sibbald et al. Increased Bacterial Burden and Infection: The Story of NERDS and STONES. ADV SKIN WOUND CARE 2006; 19: 447-61). The fluorescence image data obtained with the device can be used to characterize, spatially and spectrally, the bacterial balance and the load on the superficial and deep levels of the wounds. The device can provide non-invasive real-time guided image biopsy guidance, clinical procedure guidance, tissue characterization and can allow for image-guided treatment using conventional and emerging modalities (e.g., photodynamic therapy, PDT). The device can be used in the clinical setting and integrated into conventional clinical regimes for wound care, and may have a different role in areas of infectious diseases. It should also be borne in mind that this device can also be used for real-time analysis, monitoring and care of chronic and acute wounds in animals and pets, through conventional veterinary care.

This device can allow the evaluation of wound healing in real time for a large patient cohort base. In particular, the elderly, diabetics, immunocompromised persons and immobilized persons have a higher incidence of chronic wounds and other dermal conditions resulting from poor circulation and immobility, e.g. ex. pressure ulcers, such as sores, venous stasis ulcers and diabetic ulcers. These chronic conditions significantly increase the cost of care and reduce the patient's quality of life. As these groups increase in number, the need for advanced wound care products will increase. This device can affect patient care by allowing a cost-effective means of monitoring chronic and acute wounds in various settings, including hospitals, outpatient clinics, chronic care centers, home health care, emergency rooms and other critical areas in centers. doctors. In addition, said nursing and ambulance personnel can easily transport and use a manual and portable imaging device. The early identification of the formation of scars, which is related to the production of connective tissue and the remodeling of the wound, and bacterial infections can be detected and treated properly, something that is currently difficult. In addition, recent product developments Advanced wound care include multiple types of dressings (e.g., Films, hydrocolloids, foams, antimicrobials, alginates, non-adherent, impregnated), hydrogels, wound cleansers and debridement agents, tissue genotechnological products (e.g. eg, skin replacements, substitutes and tissue genotechnological products, such as synthetic biological tissues based on polymers and growth factors, wound cleaners, pharmacological products and physical therapies can also benefit from the device developed here, since it can allow supervision longitudinal based on images of the effectiveness of such treatments Physical therapies may include hydrotherapy, electrical stimulation, electromagnetic stimulation devices, ultraviolet therapy, hyperbaric oxygen therapy, ultrasound devices, light emitting diode (LED) / laser devices and images / wound documentation ace.

Wound tissue analysis is usually necessary to evaluate the healing of skin wounds. The percentage of granulation tissue, fibrin and wound necrosis and its change during treatment can provide useful information that can guide the treatment of the wound. Image analysis may include classification and recognition algorithms of advanced statistical patterns to identify individual pixels in the images of fluorescence wounds collected with the device based on the optical information of the wound and surrounding normal tissue. Therefore, image analysis can allow wound images to be mapped into various wound components, including the total area of the wound, epithelialization, granulation, desquamation, necrotic, hypergranulation, infection , the undercutting of the tissue and the margins of the surrounding tissue. This has the additional advantage of providing a relatively rapid determination of wound healing rates, as well as an informative guide on patient management decisions.

Figure 25 illustrates the projected administration workflow for the imaging device in a clinical wound care environment. The device can be easily integrated into routine wound assessment, diagnosis, treatment and longitudinal response monitoring, and can provide critical biological and molecular information of the wound in real time for rapid decision making during interventions adaptive

This device can be easily integrated into existing healthcare infrastructures (e.g., desktop and pocket PCs used by an increasing number of doctors or other healthcare professionals) for longitudinal cataloging of images for the treatment of wounds in patients within the conventional clinical environment. The wireless reception and transmission of the device's data capabilities can allow care monitoring and wound healing remotely through existing and future wireless telemedicine infrastructure. The device can be used to transfer essential medical data (e.g., wound status) through internet or wireless services, such as mobile phone, PDA or smartphone services, to remote sites that may allow interventions remote medical, with additional utility in military medical applications for the treatment of wounds on the battlefield. The device can allow real-time surface imaging of the wound sites to be obtained and staff at the point of care can easily carry it in clinical settings. The use of highly sensitive and cost-effective digital imaging devices available in the market, such as digital cameras, mobile phones, PDAs, laptops, tablets, webcams and smartphones, etc., as a capture or recording component of images, the device can offer image-based documentation of wound healing and monitoring the effectiveness of the treatment. In addition, this technology can also be adapted to work in "wireless" mode to allow remote medical interventions by potentially adapting it for use with high resolution digital cameras integrated into commercially available mobile phones.

By using web-based telemedicine and remote medical supervision infrastructure, the imaging device can be integrated into a "storage and shipping" concept of wound evaluation systems. In addition to providing digital images, this system can present a complete set of clinical data that meets the recommendations of the clinical practice guidelines. The device currently disclosed can be integrated into a computer-based wound evaluation system (e.g., with image analysis software) to be used by a health care center to improve existing clinical databases and support the implementation of evidence-based practice guides. Such integrated telemedicine infrastructure can be used to monitor patients at home or in long-term care facilities, who may benefit from routine supervision by qualified doctors, but currently do not have access to this care. This device can be further developed in a manual portable point of care diagnostic system, which can represent a breakthrough in the detection, supervision, treatment and prevention of the spread of infectious diseases in developed and developing countries. This knowledge can significantly improve the diagnostic tools available to professionals who treat chronic wounds in environments where quantitative cultures are inaccessible.

The device can allow digital images with optical and digital zoom capabilities (e.g., those integrated into commonly available digital imaging devices). The quality of still image or video can be in "high definition" format to achieve high spatial resolution images of the tissue surface. Images can be recorded as still / frozen images and / or in video / movie format and printed using conventional image printing protocols that require (e.g., connect via USB) or not (e.g., PictBridge) require a personal computer Image / video data can be transferred to a personal computer for data file storage and / or display and / or analysis / image manipulation. The device can also transfer data to a printer or personal computer using wired or wireless capabilities (e.g., Bluetooth). The display can be done on the screen of the manual device and / or in addition to the simultaneous display on a screen / video monitor (eg, visor helmets and glasses) using conventional video output cables. This device can display, in combination or separately, information on optical wavelength and fluorescence / reflectance intensity with spatial dimensions of the image scene to allow quantitative measurements of distances (e.g., monitor changes in the tissue morphology / topography) over time. The device can also allow the storage of digital images / video / cataloging of images and related medical data of the patient, for example, using dedicated software with image analysis capabilities and / or diagnostic algorithms.

Image analysis

Image analysis can be used together with the device to quantitatively measure fluorescence intensities and relative changes in multiple fluorescence spectra (e.g., multiplexed images) of exogenous optical molecular targeting probes in the wound and tissues surrounding normal. The biodistributions of the fluorescent probes can be determined based on the fluorescence images collected and these can be monitored over time between sessions of images of individual clinical wounds to detect changes. By determining the presence and relative changes in abundance quantitatively, using the device, of each and every one of the spectrally unique fluorescent probes, the clinical operator can determine in real time or almost in real time the state and health response and / or healing for the treatment over time of a particular wound, for example, by using a consultation table showing specific tissue, cellular and molecular signals in correlation with health, healing status and response of the wound, whose example is shown in Figure 21 (adapted from Bauer et al., Vasc & Endovasc Surg 2005, 39: 4). This may allow the doctor to determine if a wound is healing based on biological and molecular information that, otherwise, could not be possible with existing technologies. In addition, the presence and abundance of bacteria / microorganisms and their response to treatment can offer a means to adapt therapy in real time instead of incurring delays in evaluating the response with conventional bacteriological tests of wound cultures.

Image analysis techniques can be used to calibrate the initial or first images of the wound using a portable fluorescent criterion placed within the field of vision during imaging with the device. Image analysis may also allow visualization of a false or pseudocolor on a monitor to differentiate different biological components (e.g., tissues, cellular and molecular) from the wound and surrounding normal tissues, including biomarkers identified by autofluorescence. and those identified by the use of exogenous directed or non-directed directed fluorescence / absorption contrast agents.

Examples of such biomarkers are listed in Figure 22 (adapted from Brem et al. Journal of Clinical Investigation, 117: 5, 2007) and illustrated in Figure 23. In Figure 23, the diagram shows the mechanisms of wound healing in healthy people compared to people with diabetic wounds. In healthy individuals (left), the acute wound healing process is guided and maintained by integrating multiple molecular signals (e.g., in the form of cytokines and chemokines) released by keratinocytes, fibroblasts, endothelial cells, macrophages and platelets During wound-induced hypoxia, vascular endothelial growth factor (VEGF) released by macrophages, fibroblasts and epithelial cells induces phosphorylation and activation of eNOS in the bone marrow, which causes increased levels of NO, which triggers the mobilization of EPCs from the bone marrow to the circulation. For example, the chemokine SDF-1a promotes the return of these EPCs to the site of the lesion, where they participate in neovasculogenesis. In a murine model of diabetes (right), the phosphorylation of eNOS in the bone marrow is affected, which directly limits the mobilization of EPC from the bone marrow to the circulation. The expression of SDF-1a decreases in epithelial cells and myofibroblasts in the diabetic wound, which prevents EPC from returning to wounds and, therefore, limits wound healing. It has been shown that the establishment of hyperoxia in the wound tissue (e.g., through HBO therapy) activated many NOS isoforms, increased NO levels and increased mobilization of EPC to the circulation. However, local administration of SDF-1a was required to trigger the return of these cells at the wound site. These results suggest that HBO therapy combined with the administration of SDF-1a may be a potential therapeutic option to accelerate the healing of diabetic wounds alone or in combination with existing clinical protocols.

Pre-assigned color maps can be used to simultaneously display the biological components of the wound and surrounding normal tissues, including connective tissues, blood, microvascularity, bacteria, microorganisms, etc., as well as drugs / pharmacological agents. marked with fluorescence. This can allow real-time or near-real-time visualization (e.g., less than 1 minute) of the health, healing and infectious state of the wound area.

Image analysis algorithms can provide one or more of the following features:

Patient digital image management

• Integration of a variety of image acquisition devices

• Registration of all image parameters, including all exogenous fluorescence contrast agents

• Calibration settings and multiple scales

• Calculation algorithms and spectral image mixing incorporated for the quantitative determination of tissue / bacterial autofluorescence and exogenous fluorescence signals

• Convenient annotation tools

• Digital file

• Web publication

Basic image processing and analysis

• Complete package of image processing and quantitative analysis functions. The image paste algorithms will allow you to paste a series of panoramic or partially overlapping images of a wound into a single image, either in automatic or manual mode

• Easy to use measurement tools

• Intuitive configuration of processing parameters

• Convenient manual editor.

Report generation

• Powerful image report generator with professional templates that can be integrated into existing clinical report infrastructures or medical data infrastructures of telemedicine / e-health patients. Reports can be exported to PDF, Word, Excel, for example.

Large library of automated solutions

• Customized automated solutions for various areas of wound evaluation, including quantitative image analysis.

Although algorithms, techniques or image analysis software have been described, this description also extends to a computing device, a system and a method for carrying out this image analysis.

Cytoblast therapy and cancer monitoring

The device can be used for imaging and detection of cancers in humans and / or animals. The device can be used to detect cancers based on the inherent differences in fluorescence characteristics between said cancers and the surrounding normal tissues in patients. This device can also be used for the detection of image-based cancers in pets, for example, in veterinary settings.

The device can also be used as a research tool for multispectral imaging and monitoring of cancers in experimental animal models of human diseases (eg, wounds or cancers). The device can be used to detect and / or visualize the presence of cancers and track tumor growth in animal models of cancer, particularly using tumor cell lines transfected with fluorescent protein (e.g., at visible wavelength ranges and NIR)

The imaging device can be used in conjunction with existing and emerging cell therapies useful for reconditioning chronic wounds and speed healing. For this, fluorescently labeled cytoblasts can be administered at the wound site before imaging with the device. Pluripotential cytoblasts (PSCs), the precursors of all the more specialized cytoblasts, are able to differentiate into a variety of cell types, including fibroblasts, endothelial cells and keratinocytes, all of which are critical cellular components for healing. A recent report on an uncontrolled clinical trial suggests that the direct application of autologous bone marrow and its cultured cells can accelerate the healing of chronic non-healing wounds (Badiavas et al., Arch Dermatol 2003; 139 (4): 510-16 ). Given the pathophysiological abnormalities present in chronic wounds, there is a possibility that cytoblasts can reconstitute the dermal, vascular and other components necessary for optimal healing. The device can be used to visualize and track the cytoblasts marked at the site of the wound over time and determine their biodistribution and therapeutic effect. The use of agents targeting exogenous molecular fluorescence, for example, as described above, can confirm the differentiation of cytoblasts in vivo and can also help determine the wound response to this treatment.

For example, this device can be used to identify, track and / or monitor neoplastic tumor cytoblasts and cytoblasts in general (e.g., in experimental models of preclinical small animal cancers and other clinical models). An example is shown in the Figures. The device may also be useful for imaging clinical cell therapies, including the treatment of diseases using cytoblasts.

Reference is now made to Figure 18. In a), a mouse model is shown using white light. In b), the individual mouse organs are clearly seen using the fluorescence imaging device, c) shows the mouse liver photographed with the device, and no fluorescence is seen. d) shows the lungs of the mouse in white light. e) shows the lungs of the mouse photographed with the device, with neoplastic tumor cytoblasts clearly observed as bright fluorescent dots.

Referring now to Figure 19, in a), the liver of the mouse model of Figure 18 is not visible under fluorescence images. b), d) and f) show different views of the mouse lungs under white light. c), e) and g) show the corresponding view of the lungs of mice with images of the device, clearly showing the neoplastic tumor cytoblasts as bright fluorescent dots.

Figure 19H shows an example of the use of the device for the detection of nude mice with human ovarian tumors. a) White light image of virus treated and untreated control mice, showing an open abdominal cavity, b) the corresponding fluorescence image of the treated and control mice shows an orange-red fluorescence of the optically labeled virus in the tumor nodules in the mesentery (yellow arrows), in comparison to the control, c) shows an enlarged view of the mesentery, which illustrates the biodistribution of the virus's optical probe within the tumor nodules, as well as the ability to detect submillimeter tumor nodules (blue arrow), compared to the control mouse d). Note that the fluorescence of the probe can be differentiated from the autofluorescence of the intestinal background tissue. These data illustrate the potential use of the device for the response to the image treatment that includes, among others, virotherapies and cell therapies, as well as for image-guided surgical resection of fluorescent tumor samples (c; boxes) (405 nm excitation ), emission 500-550 nm (green), emission> 600 nm (red)).

Figure 19I shows an example of the use of the device for the detection / visualization in nude mice bearing colon tumors that receive a fluorescent cocktail of exogenous probes separated from targeting to green and red tumor cells after the operation. a) White light and b) corresponding multispectral fluorescence image of the open abdominal cavity showing the simultaneous detection of the green (green arrow) and red (red arrow) molecular probes, which can be analyzed with the spectral mixing software. The device can be modified to also allow obtaining endoscopic images. In this example, c) a rigid endoscopic probe was attached to the manual imaging device and d) white light and e) fluorescence images of surgically resected tissue from the mouse were obtained in image a, b). These data suggest the use of the device with endoscopic probe accessories for real-time portable endoscopic fluorescence imaging in vivo in human and veterinary patients for a variety of treatment detection, diagnosis or monitoring applications (based on research and clinical) , f) the device (eg, with endoscopic capabilities) may be able to obtain fluorescence images of multiple spectrally unique "probes" that can be used in vivo (405 nm excitation; 490-550 nm emission channels and > 600 nm).

This device can be used for multispectral imaging and detection of cancers in humans and animals. This device can also be used to detect cancers based on the inherent differences in fluorescence characteristics between said cancers and the surrounding normal tissues in patients. This device can also be used for the detection of image-based cancers in animals such as pets or livestock, for example, in veterinary settings.

This device may also be suitable as a research tool for multispectral imaging and monitoring of cancers in experimental animal models of human diseases (eg, wounds and cancers). The device can be used to detect and / or visualize the presence of cancers and can be used to track tumor growth in animal models of cancer, particularly using tumor cell lines transfected with fluorescent proteins (e.g., in length ranges visible wave and NIR).

Image guidance

The device may also be useful for providing a fluorescent imaging guide, for example in surgical procedures, even without the use of dyes or markers. Certain tissues and / or organs may have different fluorescent spectra (eg, endogenous fluorescence) when viewed with the imaging device, or for example under certain conditions of excitation light.

Figure 20 demonstrates the usefulness of the device for fluorescence imaging assisted surgery. With the help of fluorescence images using the device, different organs of a mouse model can be distinguished more clearly than under white light. b, c and g show the murine model under white light. a, d-f and h-j show the murine model as shown with the device.

Figure 20B shows an example of the use of the device to obtain images of small animal models. In this case, the dorsal fold window chamber of the mouse skin is visualized under white light (a, c) and fluorescence (b, d). Note the high resolution white light and fluorescence images obtained by the device. The feet and face have a bright fluorescent red color due to the endogenous autofluorescence of the cage bed and food powder materials. (405 nm excitation; 490-550 nm and> 600 nm emission channels).

Skin made by bioengineering

Several skin products made by bioengineering or skin equivalents are commercially available for the treatment of acute and chronic wounds, as well as for burns. These have been developed and tested on human wounds. Skin equivalents may contain living cells, such as fibroblasts or keratinocytes, or both, while others are made of acellular materials or living cell extracts (Phillips. J Dermatol Surg Oncol 1993; 19 (8): 794-800). The clinical effect of these constructs is 15-20% better than conventional "control" therapy, but there is a debate about what constitutes appropriate control. Bioengineered skin can function by providing live cells that are known as "intelligent material" because they are able to adapt to their environment. There is evidence that some of these living constructs may release growth factors and cytokines (Falanga et al. J Invest Dermatol 2002; 119 (3): 653-60). Exogenous fluorescent molecular agents may be used together with such skin substitutes to determine the integrity of the graft, as well as the biological response of the wound to therapy. The cure of full thickness skin defects may require extensive synthesis and remodeling of the dermal and epidermal components. Fibroblasts play an important role in this process and are incorporated into the latest generation of artificial dermal substitutes.

The imaging device described herein can be used to determine the fate of sown fibroblasts in the skin substitute and the influence of sown fibroblasts in cell migration and degradation of the dermal substitute after site transplantation can be determined. of the wound. Wounds can be treated with dermal substitutes seeded with autologous fibroblasts or acellular substitutes. The seeded fibroblasts, labeled with a fluorescent cell marker, can then be detected in the wounds with a fluorescence imaging device and then quantitatively evaluated using image analysis, for example, as described above.

Therapeutic agents based on polymers

There are a number of commercially available medical polymer products for wound care. For example, Rimon Therapeutics produces Theramers ™ (www.rimontherapeutics.com) which are medical polymers that have biological activity in themselves, without the use of drugs. Rimon Therapeutics produces the following wound care products, which can be converted to single fluorescent when excited with 405 nm excitation light: Angiogenic Theramer ™, which induces the development of new blood vessels (i.e. angiogenesis) in wounds or other ischemic tissue; MI Theramer ™, which inhibits the activity of matrix metalloproteases (MMPs), a ubiquitous group of enzymes that are involved in many conditions in which the tissue is weakened or destroyed; AM Theramer ™, a thermoplastic that destroys gram and large bacteria - without damaging mammalian cells; and ThermaGel ™, a polymer that changes from liquid to strong gel reversibly around body temperature. Each can be fluorescent by the addition of fluorescent dyes or fluorescent nanoparticles selected to be excited, for example, in 405 nm light with a longer wavelength fluorescence emission.

When using the imaging device, the application of said fluorescent polymer agents can be guided by real-time fluorescent images. This may allow the Theramer agent to be administered / applied accurately (e.g., topically) at the site of the wound. After application of the agent to the wound, the fluorescent imaging device can be used to quantitatively determine the therapeutic effects of Theramers on the wound, as well as to track their biodistribution in the wound over time, in alive and not invasively. It may also be possible to add a molecular beacon, possibly with another fluorescent emission wavelength, to the Theramer ™ MI that can emit fluorescence in the presence of wound enzymes (e.g., MMPs), and this may indicate in real time the wound response to the MI Theramer ™. It may be possible to use a fluorescence emission for the image-guided Theramer application at the wound site and a different fluorescence emission for monitoring the therapeutic response and other fluorescence emissions for other measurements. The relative effectiveness of MMP inhibition and antimicrobial treatments can be determined simultaneously over time. Using image analysis, real-time comparison of the changes in fluorescence of these signals in the wound may be possible. This prints a quantitative aspect to the device, and prints its clinical utility.

It should be noted that other bio-safe fluorescence agents can be added to the following materials that are currently used for wound care. The fluorescent material can then be visualized and monitored using the device.

• Wet wound dressings: This provides a moist and conducive environment to improve healing rates compared to traditional dressings. The main consumer base that manufacturers are targeting for these dressings is people over 65, who suffer chronic injuries such as pressure ulcers and venous stasis ulcers. Those with diabetes and as a result, developed ulcers are part of the target population.

• Hydrogels: this adds moisture to dry wounds, creating a suitable environment for faster healing. Its additional feature is that they can be used on infected wounds. These are also designed for dry to slightly exudative wounds.

• Hydrocolloid dressings: Hydrocolloids seal the wound bed and prevent moisture loss. They form a gel by absorbing exudates to provide a moist healing environment. These are used for mild to moderately exudative wounds without infection.

• Alginate dressings: absorb wound exudates to form a gel that provides a moist environment for healing. They are mainly used for highly exudative wounds.

• Foam dressings: these absorb the drainage of the wound and maintain a moist surface of the wound, allowing an environment conducive to wound healing. They are used in moderately exudative wounds.

• Transparent film dressing: they are not absorbent, but allow moisture vapor permeability, thus ensuring a wet wound surface. They are intended for dry or slightly exudative wounds. Examples include transparent film dressings based on alginate foam.

• Antimicrobials: provide an antibacterial action to disinfect the wound. Of particular interest is the use of nanocrystalline silver dressings. The biological load, particularly the proteases and accumulated toxins released by bacteria that hinder healing and cause pain and exudation, is significantly reduced with prolonged release of silver.

• Active wound dressings: these include highly evolved tissue engineering products. Biomaterials and skin substitutes are included in this category; These are composed entirely of biopolymers such as hyaluronic acid and collagen or biopolymers together with synthetic polymers such as nylon. These dressings actively promote wound healing by interacting directly or indirectly with wound tissues. Skin substitutes are bioengineering devices that personify the structure and function of the skin.

• Hyaluronic acid: this is a natural component of the extracellular matrix and plays an important role in granular tissue formation, reepithelialization and remodeling. It provides hydration to the skin and acts as an absorbent.

Other products for wound care that can be displayed using the disclosed device include Theramers, gels containing silver (p. G., Hydrogels), artificial skin, stem cells ^to D ^d, anti matrix metalloproteinases and hyaluronic acid. Fluorescent agents can be added to other products to allow imaging using the device. In some cases, products may already be luminescent and do not require the addition of fluorescent agents.

The device can also be used to monitor the effects of such treatments over time.

Application for food products

The imaging device may also be useful for monitoring food products (eg, meat products) for contamination. This may be useful, for example, in the preparation of food / animal products in the meat, poultry, dairy, fisheries and agricultural industries. The device can be used as part of an integrated multidisciplinary approach to laboratory analytical services within this sector, which can provide capabilities that include image-based contamination detection and a guide to obtaining samples for testing. The device can be used for the detection, identification and real-time monitoring of the level of contamination / adulteration of bacterial and microbial meat from other food products. It can be used for monitoring bacterial contamination in the food processing plant environment and, therefore, can provide an image-based method to determine food safety and quality. In embodiments where the device is manual, compact and portable, the imaging device may be useful in food preparation areas to determine the safety of food products against bacterial / microbial contamination. The device can also be used for the relatively rapid detection and analysis of bacteria / microbes in meat samples (and on preparation surfaces) collected or sampled, for example, as part of the regulated food quality and safety inspection process. , during processing and in finished food products. This device can be used in the meat, horticulture and aquaculture industries to implement food safety inspection / detection procedures that meet food safety and quality requirements. The device can be used to detect contaminants in food, for example, contaminants found in the meat, poultry, dairy and fisheries industries. This technology can be useful as a fecal contaminant detection system, since fecal bacteria produce porphyrins that the device can easily detect.

Accurate detection and identification of foodborne pathogens, such as Listeria monocytogenes (LM), in food samples and in processing lines can be critical both to ensure food quality and to track outbreaks of bacterial pathogens in the food supply. The current detection methods used in the production and processing facilities of Foods are generally based on multiple random sampling of the equipment surface (e.g., sampling) and subsequent molecular-based diagnostic tests (e.g., real-time polymerase chain reaction, RT-PCR ) that can provide quantitative confirmation of the presence of LM, usually within 24-72 hours. However, due to time and cost constraints, generally only randomly selected areas of a given food production facility are tested for pathogen contamination at a time, and the significant potential of a subsample during sampling of "first pass" surface of the equipment may not detect pathogens that cause catastrophic health and economic consequences. In addition, the inability to i) quickly sample all areas of the surface during "first pass" sampling to identify areas with high probability of infection, ii) to visually document this initial systematic detection process (eg, no There are imaging methods available to date), iii) the delay in obtaining laboratory results, iv) the high costs associated with current methods, and v) most importantly, the possibility that they will not be detected Mortal pathogen infections have intensified efforts to improve the early and accurate detection of foodborne pathogens in a cost-effective manner.

The device may be useful for providing a relatively fast and accurate way to detect such pathogens. The device can be used with a multicolored fluorescence probe "cocktail" assay (eg, a combination of two or more contrast agents) that can unequivocally identify (and make visible) only viable Listeria monocytogenes of other species of Listeria using highly specific gene probe technology. This can allow the specific detection of live LM in real time, potentially minimizing the need for conventional enrichment methods that require a lot of time. This method can also be expanded to include the detection of other pathogens of interest, including Enterobacter sakazakii, Camylobacter species (C. coli, C. jejuni and C. tari), coliform bacteria and bacteria of the E. coli species (including strains of Escherichia coli negative lactose and indoles), Salmonella, all the bacteria belonging to the Staphylococcus aureus species and separately all the bacteria belonging to the genus Staphylococcus and Pseudomonas aeguginosa. Other bacteria can be detected by selecting a suitable probe or combination of probes. For example, a combination of two or more contrast agents can be designed to be specific for certain bacteria, and can result in a unique detectable fluorescent badge when an image is taken using the imaging device.

The imaging device can be used (eg, when combined with specific contrast agents for exogenous bacteria applied, including a multidian probe or a combination of probes) for the relatively rapid systematic detection of "first pass" of food preparation and handling surfaces for specific sampling and microbiological testing. This device can allow relatively fast image-based surveillance of any surface of equipment and food products and can capture the fluorescence badge of foodborne bacteria / pathogens in real time. The device can be used in combination with, for example, a "cocktail" assay of multi-colored fluorescence probes (and combinations thereof) that can unequivocally identify (and make visible) only viable Listeria monocytogenes from other Listeria species using highly gene specific probe technology, as described above. Said "cocktail" of probes can be designed to specifically target certain pathogens based on a specific combination of probes that are known to be sensitive to such pathogens and that are known to give a characteristic fluorescence response. In addition to the detection of said pathogens, the device may allow differentiation of the presence and / or location of different strains, depending on their different distinctive fluorescence response.

Figure 26 shows an example of the use of the imaging device for real-time examination of meat products in the food supply. In this case, a) white light and b) the corresponding autofluorescence image of a piece of pork shows the difference between various tissues, including bone and tendon (white arrow), fat and muscle, c) white light and b) the corresponding autofluorescence image of a "cutting edge" of the bone, in which the cartilage (blue arrow) has a bright green color under fluorescent light due to collagen autofluorescence, while several types of Internal tissues of the bone, including the bone marrow (red arrow) can be differentiated using fluorescence. The last observation may further suggest the use of the manual imaging optical device for real-time fluorescence imaging guidance during orthopedic surgery in human and veterinary patients, as discussed above. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)).

Figure 27 shows another example of the use of the imaging device for real-time examination of meat products in the food supply. In this case, a) white light and b) corresponding autofluorescence images of a piece of pork that has been kept for 2 days at 37 ° C. The autofluorescence image shows the presence of mixed bacterial contamination on the surface of the meat (areas of red fluorescence; yellow arrows) that include, for example, Staphylococcus aureus and E. Coli. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)).

Surface contamination

The imaging device may be useful for the detection of surface contamination, such as the detection of "bacterial surface contamination" in healthcare settings. This device can be use to detect and visualize the presence of bacteria / microbes and other pathogens on a variety of surfaces / materials / instruments (particularly those related to surgery) in hospitals, chronic care centers and nursing homes, where there is contamination. It is the main source of infection. The device can be used together with the detection, identification and conventional enumeration of indicator organisms and pathogen strategies.

Figure 28 shows an example of the use of the imaging device for real-time examination of soil and algae samples, in an example of environmental sampling / detection of contaminants. A) White light and b) Corresponding autofluorescence images of a Petri dish containing a sample of soil and mineral. c) An example of the imaging device used to detect fluorescent earth contaminants / hazardous materials. In this case, for example, a fluorescein-labeled liquid was added to the ground before the fluorescence image to illustrate the potential use of the imaging device for the detection and monitoring of environmental pollutants and contaminants, d) an example of the imaging device used to obtain white light and e) autofluorescence images of a culture of green algae grown in laboratory conditions, illustrating the potential usefulness of the imaging device for real-time monitoring of water conditions based on fluorescence imaging (e.g., drinking water purification / safety tests, or algal growth in large-scale production plants). As an example of the imaging device used to detect diseases in plants, f) shows a white light image of a common household plant, while g) shows the corresponding autofluorescence image of a fungal infection that has a bright green color ( yellow arrows) that affects plant leaves, compared to healthy leaf tissue that has a bright reddish brown color. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)). Therefore, the device may be useful for obtaining images of plant materials.

Figure 28B shows an example of the use of the imaging device used for the detection of hidden contamination with white light of biological fluids in public and private environments, a) white light and bc) corresponding autofluorescence of the biological fluids that contaminate a Toilet and a bathroom vanity top. These data suggest that the imaging device can be used to detect surface contamination by samples / biological / infectious fluids potentially dangerous for sampling, cleaning or image-guided monitoring. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)).

Figure 28C shows an example of the use of the device for detecting bacterial contamination of surgical instruments (b; green arrow) using fluorescence images. (405 nm excitation channels; 490-550 nm and> 600 nm emission).

Forensic Uses

The use of the imaging device to identify contaminants and surface targets may be useful in forensic applications. For example, the device may be useful for the forensic detection of latent fingerprints and biological fluids on non-biological surfaces. The device can offer a relatively inexpensive, compact and portable means of obtaining digital images (eg, with white light, fluorescence and / or reflectance) of latent fingerprints and biological fluids, and other substances of forensic interest. The former can be made fluorescent using commercially available fingerprint fluorescence dyes, and the latter can be detected using the autofluorescence of exogenously applied "directed" fluorescent fluids or coloring agents (such as Luminol). Images can be recorded digitally. The device can also be used during autopsy procedures to detect bruises.

Figure 29 shows an example of the use of the imaging device for real-time fluorescence detection of liquid leaks using an exogenous fluorescent tracer dye. a) White light image of a typical tap, b) corresponding fluorescence image (showing the presence of leaking fluid (with added fluorescence dye), and composite image of white light and fluorescence. Note that leakage ( in this example, water) is not visible with white light, but is easily detected using fluorescence.This data suggests that the imaging device may be useful for image formation tracking and relatively fluid / fluid leak detection. fast (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)).

Figure 30 shows an example of the use of the imaging device for real-time fluorescence detection of surface contaminants). a) white light image of a typical laboratory bench surface and b) an area to be visualized with the imaging device, c) fluorescence images can be used to detect contaminants that are not easily visualized with light white (a, b). The imaging device can also be used to detect latent fingerprints, for example, using a fluorescent dye to improve the ridges of fingerprints on a table surface. This can be done, for example, by including a fluorescent dye combined with superglue (e.g., cyanoacrylate) to develop a contrast of fingerprints against the bottom surfaces. Fluorescent dyes of Far infrared and near infrared to reduce the autofluorescent background potential. These data suggest the use of the imaging device for relatively rapid detection based on images of non-biological and biological contaminants, as well as fingerprints, for example, in forensic applications. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)).

The device can also be useful in counterfeit applications. Figure 31 shows an example of the imaging device used to obtain images of common currency (in this example, a Canadian $ 20 bill) in a) white light and b, c) autofluorescence modes. Invisible under white light (a), special measures can be observed against counterfeiting under fluorescence: that is, the embedded fluorescence fibers (b) and the embedded watermark of banknotes (c) can be spectrally distinguished (arrows ). These data suggest that the device can be used for counterfeiting purposes. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)).

Cataloging

The imaging device can allow the cataloging of fluorescent-based animals, such as laboratory animals. Figure 32 shows an example of the use of the imaging device for real-time fluorescence detection of "barcode" identification tags for laboratory animals. The figure shows a) a white light image of a typical laboratory rat and b) a fluorescence image of the rat labeled with a fluorescent bar code. The use of multiple fluorescent dyes / colors in combination with bar code patterns / bars can be used for "multiplexed cataloging" of animals, for example, for longitudinal research studies. These data suggest the use of the imaging device for bar code cataloging based on relatively fast high performance images of laboratory animals for use in c) colonies of "pathogen containment" animals in research laboratories and for genotyping of animals (eg, transgenic animals, box in c), for example. (405 nm excitation, 500-550 nm emission (green),> 600 nm emission (red)). The device can also be used to obtain images of fluorescence-based barcodes or other coding systems in other applications, such as inventory tracking and point-of-sale tracking.

Kits for a device

The imaging device may be provided in a kit, for example, including the device and a fluorescent contrast agent. The contrast agent may be one or more of those described above. For example, the contrast agent may be to mark a biomarker on a wound, in which the kit is for wound monitoring applications.

Figure 33 shows an example of a kit that includes the imaging device, a) shows the handle and the touch display screen, and b) shows the external housing and the excitation light sources. The imaging device can be used to scan the body surface of human and veterinary patients for evaluation of image-based wounds, or for non-wound imaging applications. The device and any accessories (e.g., battery / power supplies), potential exogenous fluorescence contrast agents, etc.) can be conveniently placed in rigid containers for transport in clinical and non-clinical settings (including sites remote, nursing home and research laboratory environments).

Cosmetic or dermatological uses

The imaging device can also be used to obtain images of cosmetic or dermatological products.

Figure 34 shows an example of the use of the device to obtain images of cosmetic products. For example, four commercially available cosmetic creams are shown in a) white light and b) fluorescence imaging modes, which show the fluorescence contrast between the creams and the background skin. These data illustrate the potential use of the manual imaging device for use in the presence image and the possible biological effects of cosmetics (e.g., skin rehydration, collagen remodeling, sunburn damage repair , skin exfoliation) and dermatological agents or drugs (excitation channels of 405 nm; emission of 490-550 nm and> 600 nm)).

The imaging device can be used in white light and fluorescence modes to improve the administration of these treatments, as well as to monitor their effectiveness over time in a non-invasive and quantitative manner. The device can be used in combination with other imaging modalities, for example, thermal imaging methods, among others.

This device can also be used to test antibacterial agents, antibiotics or disinfectants. The fluorescence images provided by this device can be used, for example, in combination with white light images, to quantitatively detect the effectiveness of the treatments. Pharmacists in bacterial cultures and other model systems, during the discovery, optimization and evaluation of drugs, for example, for the treatment of wounds.

All examples and embodiments described herein are for illustration purposes only and are not intended to be limiting. One skilled in the art will understand that other variations are possible.

Claims (15)

A device for imaging and fluorescence monitoring of a target (10), comprising: a light source (5) that emits a light to illuminate the target, the emitted light includes at least one wavelength or band wavelength that causes the fluorescence of at least one biomarker associated with the target; an optical filter holder (3) configured to accommodate one or more optical filters; an electrical supply (19); a digital image acquisition device (1) that has a lens (2) and is configured to acquire a fluorescence image of the target secured inside the housing, in which the digital image acquisition device is a camera digital, a video recorder, a camcorder, a mobile phone with a built-in digital camera, a smartphone with a digital camera, a personal digital assistant or a webcam; Y a housing (20) that houses all the components of the device in an entity and comprises a means for fixing the digital image acquisition device in the housing, in which the housing is configured to be manual, compact and portable. 2. The device of claim 1, wherein the target is selected from the group consisting of: a surgical field, a wound, a tumor, an organ, a skin target, a biological target, a non-biological target, a food product, a plant material, an oral target, an otolaryngological target, an ocular target, a genital target, and an anal target. 3. The device of any one of claims 1 or 2, wherein the at least one biomarker is selected from the group consisting of bacteria, fungi, yeasts, spores, viruses, microbes, parasites, connective tissues, tissue components, exudates, pH, blood vessels, nicotinamide adenine dinucleotide reduced (NADH), flavin adenine dinucleotide (FAD), microorganisms, vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), epithelial growth factor, membrane antigen epithelial cells (ECMA), hypoxia-inducible factor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin, fibroblast growth factor, transforming growth factors (TGF), fibroblast activation protein (FAP) ), tissue inhibitors of metalloproteinases (TIMPs), nitric oxide synthase (NOS), inducible and endothelial NOS, cell lysosomes, macrophages, neutrophils, lymphocytes, growth factor hepatocyte (HGF), antineuropeptide, neutral endopeptidases (NEP), granulocyte and macrophage colony stimulating factor (GM-CSF), neutrophil elastases, cathepsins, arginase, fibroblasts, endothelial cells and keratinocytes, keratinocyte growth factor (KG ), macrophage inflammatory protein-2 (MIP-2), macrophage inflammatory protein-2 (MIP-2), and macrophage chemoattractant protein-1 (MCP-1), polymorphonuclear neutrophils (PMN), macrophages, myofibroblasts, interleukin -1 (IL-1), tumor necrosis factor (TNF), nitric oxide (NO), c-myc, betacatenin, endothelial progenitor cells (EPCs), matrix metalloproteinases (MMPs) and m Mp inhibitors. 4. The device of any one of claims 1-3, wherein the emitted light includes wavelengths from about 400 nm to about 450 nm. 5. The device of any one of claims 1-4, wherein the emitted light includes wavelength bands selected from the ranges of about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 600 nm at about 650 nm, about 650 nm at about 700 nm, about 700 nm at about 750 nm, and combinations thereof. 6. A kit for imaging and fluorescence monitoring of a target comprising: the device of any one of claims 1 to 5; Y a fluorescent contrast agent for marking the biomarker of the target with a wavelength or band of fluorescent wavelength detectable by the device. 7. The kit of claim 6, wherein the biomarker is a bacterium, and the contrast agent is aminolevulinic acid (ALA) or PpIX. 8. The kit of claim 6, wherein the contrast agent is selected from the group consisting of: fluorescent dyes, chromogenic dyes, quantum dots (QDots), molecular beacons, nanoparticles having fluorescent agents and dispersion nanoparticles or absorption. 9. A method for imaging and fluorescence monitoring using the device of any one of claims 1 to 5 or the kit of any of claims 6 to 8 of a target comprising: lighting the target with the light source Of the device; Y Fluorescence detection with the digital image acquisition device. 10. The method of claim 9, further comprising labeling a biomarker selected on the target with at least one fluorescent contrast agent. 11. The method of claim 10, wherein the contrast agent is aminolevulinic acid (ALA). 12. The method of claim 10, wherein the contrast agent is selected from the group consisting of fluorescent molecules, chromogenic dyes, quantum dots (QDots), molecular beacons, nanoparticles having fluorescent agents and dispersion or absorption nanoparticles . 13. The method of any one of claims 10-12, comprising labeling the selected biomarker on the target with a combination of two or more contrast agents, in which the combination is specific for a selected biomarker. 14. The method of any one of claims 9-13 in combination with an additional imaging technique. 15. The method of claim 14, wherein the imaging technique is selected from the group consisting of: thermal imaging, ultrasound, white light photography and optical devices.